Cloning of a human kidney cDNA with similarity to the sodium-glucose cotransporter REBECCA G. WELLS, ERNEST M. WRIGHT,
ANA M. PAJOR, AND MATTHIAS
YOSHIKATSU A. HEDIGER
KAN.AI,
ERIC
TURK,
Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Department of Physiology, School of Medicine, University of California, Los Angeles, California 90024 Wells, Rebecca G., Ana M. Pajor, Yoshikatsu Kanai, Eric Turk, Ernest M. Wright, and Matthias A. Hediger. Cloning of a human kidney cDNA with similarity to the sodium-glucosecotransporter. Am. J. Physiol. 263 (Renal Fhid Electrolyte Physiol. 32): F459-F465, 1992.-We have usedlowstringency screening with the human intestinal Na+-glucose cotransporter SGLTl to isolate a 2,271-nucleotide cDNA (Hul4) from human kidney. This clone, which encodesa 672residue protein, is 59% identical at the amino acid level to SGLTl and has a similar number and arrangementof predicted membrane-spanningregions.It alsosharessignificant sequence identity with other Na+-coupled transporters. Northern blot analysissuggestsstrong expressionof Hu14 in kidney, but, unlike SGLTl, no significant expression in intestine. We have been unable to demonstrate definitive transport of any of a number of substrates (including amino acids, sugars, nucleosides, and vitamins) into Hu14 cRNA-injected Xenopus oocytes, although sequenceconservation makesit likely that Hul4 representsanother member of the Na+ cotransporter family, possibly a secondNa+-glucosecotransporter. glucosetransport; small intestine
and kidney proximal tubule, glucose transport across epithelial cells proceeds via Na+-glucose cotransporters in the brush-border membrane and facilitated glucose transporters in the basolateral membrane. A brush-border Na+-glucose cotransporter (SGLTl) was previously isolated from rabbit (9) and human (11) small intestine, the LLC-PK1 renal cell line (20)) and rabbit (3, 18) and rat kidney cortex (R. Wells, unpublished results). A mutation at position 28 from Asp to Asn results in the human inherited disease glucose-galactose malabsorption (23). Efficient substrate transport in the kidney is provided by low- and high-affinity transporters arranged in series along the tubule. SGLTl from rabbit has the kinetic characteristics of a high-affinity transporter with a K, for methyl-cu-D-glucopyranoside (cu-MeGlc) of 110 PM and a Na+-to-glucose coupling ratio of 2:1 (13). SGLTl is probably the only Na+-glucose cotransporter expressed in the small intestine (13). There is evidence, however, from the studies of Turner and Moran (24) using rabbit renal proximal tubule brush-border membranes and from Barfuss and Schafer (1) using perfused proximal tubule segments for a second, low-affinity Na+-glucose cotransporter in kidney outer cortex with a Na+-to-glucose ratio of 1:l. The existence of a second Na+-glucose cotransporter in human kidney is supported by the observation of Elsas and Rosenberg (7) that renal but not intestinal glucose absorption is impaired in renal glycosuria, an autosomal recessive inborn error of transport. Several recently cloned Na+ cotransporters show significant similarity to SGLTI and suggest that there is a
IN THE SMALL
INTESTINE
0363-6127/92
$2.00
Copyright
family of such transporters: the Escherichia coli Na+proline (putP; see Ref. 19) and Na+-pantothenic acid (panF; see Ref. 14) transporters, the rabbit Na+-uridine cotransporter SNSTl (22), and the Na+-myo-inositol cotransporter (SMITl; see Ref. 16). The eukaryotic y-aminobutyric acid (8), serotonin (l2), and norepinephrine (21) Na+- and Cl--dependent transporters and the E. coli glutamate (4) and rabbit kidney phosphate (27) Na+-dependent transporters have recently been cloned, but have no significant sequence similarity to SGLTl. At present, there is no information available about the molecular basis of intestinal or renal Na+-coupled organic solute transporters other than SGLTl (9), SNSTl (22) and SMITl (16). In the present paper, cDNA libraries prepared from human kidney were screened at low stringency with a rabbit SGLTl probe to search for additional Na+ cotransporter-like sequences. METHODS
Cloneswere isolatedfrom the following human kidney cortex cDNA libraries: Library 1 (nonamplified, in X-Zap) prepared in our laboratories using human kidney tissue provided by Jill Norman, UCLA Schoolof Medicine, and library 2 (amplified, in A-gtl0) obtained from Graeme Bell, University of Chicago (2). Approximately 300,000cloneswere screenedfrom each library. Library 1 wasscreenedat 37°C with the gel-purified, 32P-labeled (T7QuickPrime) rabbit SGLTI Eco RI insert (9). The hybridization solution contained 5~ standard sodium citrate (SSC), 3~ Denhardt’s solution, 0.2% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 50% formamide, 0.01% antifoam B (Sigma), 0.1 mg/ml yeast RNA (Sigma, phenol extracted), 0.2 mg/ml denatured calf thymus DNA (Lofstrand), 2.5 mM sodium pyrophosphate, and 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5. The filters were washedin 0.1~ SSC-0.1% SDS at 50°C. The following two cloneswith inserts larger than 1.5 kb were selectedfor further analysis:K7 (1.8 kb) and K15 (2.5 kb). Clone K15 was used as a probe to screen library 2. The screeningconditions were the sameasfor the first library except that the filters were washedat higher stringency (0.1X SSC-0.1% SDS at 65°C). Multiple clones were selected, including Hu5 (2.4 kb), Hull0 (2.6 kb), and Hu14’ (2.4 kb). Both strands of clone K15, the 5’- and 3’-ends of Hu5, HulO, and Hu14, and an internal region of Hu14 were sequenced.K15 was subclonedinto Ml3 mp19 and sequencedby the dideoxy-sequencing method using 35S-dATP. Isolated h-phagecDNA inserts from Hu5, HulO, and Hu14 were subclonedinto pBluescript (Stratagene) and used for double-stranding sequencing with a polymerase dideoxysequencingkit (Pharmacia). Synthetic oligonucleotideprimers were usedto complete sequencing. A computer-basedsearchof the Genbank, EMBL, Swissprot, and Pir databaseswasperformed, and sequences were alignedas l The GenBank
nucleotide sequence of clone Hu14 data base (accession no. M95549).
0 1992 the American
Physiological
Society
has been deposited
in the F459
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
1 1 SGLTl:
10
I
20
MDSSTWSPKTTAVTRPVETHE
R
30
s
A
v
I
I
V
Hu14: MEEHTEAGSAPEMGAQKALIDNPADILVIAAYFL GGGGGCAGATCCTGGGGAGAATGGAGGAGCACACAGAGGCAGGCTCGGCACCAGAGATGGGGGCCCAGAAGGCCCTGATTGACAATCCTGCTGACATCCTAGTCATTGCTGCATATTTCC 20
40
40 V
M
60
1
A
A
80
50 F
100
60
s
70
F
I
LVIGVGLWSMCRTNRGTVGGYFLAGRSMVWWPVGASLFAS TGCTGGTCATTGGCGTTGGCTTGTGGTCCATGTGCAGAACCAACAGAGGCACTGTGGGCGGCTACTTCCTGGCAGGACGCAGCATGGTGTGGTGGCCGGTTGGGGCCTCTCTCTTCGCCA 140
160
180
200
I
220
G
v
L
v
v
V
NIGSGHFVGLAGTGAASGLAVAGFEWNALFVVLLLGWLFA GCAACATCGGCAGTGGCCACTTTGTGGGCCTGGCAGGGACTGGCGCTGCAAGTGGCTTGGCTGTTGCTGGATTCGAGTGGAATGCGCTCTTCGTGGTGCTGCTACTGGGCTGGCTGTTTG 260
280
120
I
I
300
I
K
320
130
V
340
140
E
Q
Q
150
v
L
L
PVYLTAGVITMPQYLRKRFGGRRIRLYLSVLSLFLYIFTK CACCCGTGTACCTGACAGCGGGGGTCATCACGATGCCACAGTACCTGCGCAAGCGCTTCGGCGGCCGCCGCATCCGCCTCTACCTGTCTGTGCTCTCCCTTTTCCTGTACATCTTCACCA 380 400
420
160
h70
A I I N L ISVDMFSGAVFIQQALGWNIYASVIALLGITMIYTVTGGL AGATCTCAGTGGACATGTTCTCCGGAGCTGTATTCATCCAGCAGGCTCTGGGCTGGAACATCTATGCCTCCGTCATCGCGCTTCTGGGCATCACCATGATTTACACGGTGACAGGAGGGC 500
v
I
440
L
L
520
Y
L
V
I
L
A
I
V
s
19d
F'L
A
540
M
460
4 180
I
A
560
L
T
580
F
D
A
F
M
E
M
AALMYTDTVQTFVILGGACILMGYAFHEVGGYSGLFDKYL TGGCCGCGCTGATGTACACGGACACGGTACAGACCTTCGTCATTCTGGGGGGCGCCTGCATCCTCATGGGTTACGCCTTCCACGAGGTGGGCGGGTATTCGGGTCTCTTCGACAAATACC 620
640
660.
240
I
K
P
680
250
T
v
s
-
-
-DGNTTFQEK
700
260 A
T
1270
I
F
F
D
L
GAATSLTVSEDPAVGNISSFCYRPRPDSYHLLRHPVTGDL TGGGAGCAGCGACTTCGCTGACGGTGTCCGAGGATCCAGCCGTGGGAAACATCTCCAGCTTCTGCTATCGACCCCGGCCCGACTCCTACCACCTGCTCCGGCACCCCGTGACCGGGGATC 740
760
780
800
820
6
29d
280 G
F
I
F
M
s
L
T
300
L
l-
310
T
S
A
N
M
S
V
G
F
F
PWPALLLGLTIVSGWYWCSDQVIVQRCLAGKSLTHIKAGC TGCCGTGGCCCGCGCTGCTCCTCGGACTCACAATCGTCTCGGGCTGGTACTGGTGCAGCGACCAGGTC~TCGTGCAGCGCTGCCTGGCCGGGAAGAGCCTGACCCACATCAAGGCGGGCT 860
880
900
920
940
7 320
330
7
340
350 T
ILCGYLKLTPMFLMVMPGMI
F
KI
s
K
Y
SRILYPDEVACVVPEVCRRV
GC;TCCTGTGTGGGTACCTGAAGCTGACGCCCATGTTTCTCATGGTCATGCCAGGCATGATCAGCCGCATTCTGTACCCAGACGAGGTGGCGTGCGTGGTGCCTGAGGTGTGCAGGf~CG 980 1000
1020
1040
1060
c.--370
360 K
T
'380
T AYPRLVVKLMPNGLRGLMLAVMLAALMSSL
CGTEVGCSNI
390 S
E
TGTGCGGCACGGAGGTGGGCTGCTCCAACATCGCCTACCCGCGGCTCGTCGTGAAGCTCATGCCCAACGGTCTGCGCGGACTCATGCTGGCGGTCATGCTGGCCGCGCTCATGTCCTCGC 1120 1100
1140
S
1160
Fig. 1. Nucleotide sequence and deduced amino acid sequence of Hu14-K15. Residues that are different in human SGLTI are shown above amino acid sequence. Brackets indicate predicted membrane-spanning regions. * First stop codon (TAA).
1180 9
40d
410
420 A
A
S
I
'
430
K V RLR~RA~~~,,: ~GRL:: ~~~
FNSSSTLFTMDIYT
TGGCCTCCATCTTCAACAGCAGCAGCACGCTCTTCACCATGGACATCTACACGCGCCTGCGGCCACGCGCCGGCGACCGCGAGCTGCTGCTGGTGGGACGGCTCTGGGTGGTGTTCATCG 1220 1240
1260
1280
1300 10
440 G 1 VVSVAWLPVVQAAQGGQL
I
I
V
450
I
S
'
460
S
S
I
470 T
I
G
A
L
FDYIQAVSSYLAPPVSAVFVLA
TGGTAGTGTCGGTGGCCTGGCTTCCCGTGGTGCAGGCGGCACAGGGCGGGCAGCTCTTCGATTACATCCAGGCAGTCTCTAGCTACCTGGCACCGCCCGTGTCCGCCGTCTTCGTGCTGG 1340 1360
1400
1380
1420
10A ' I
W
r
480
K
500
490
I
L
P
LFVPRVNEQGAFWGLIGGLLMGLARLIPEFSFGSGSCVQP CGCTCTTCGTGCCGCGCGTTAATGAGCAGGGCGCCTTCTGGGGACTCATCGGGGGCCTGCTGATGGGCCTGGCACGCCTGATTCCCGAGTTCTCCTTCGGCTCGGGCAGCTGTGTGCAGC 1460 1480
I
r
M
s
1500
T
A
510
Y
T
1520
1540
11 N
-,._._. $
A
r
P
T A
!!zr--I
54d
530
I
I
-
A I FCSGLLTLTVSLCTAPIPRK
FLCGVHYLYFAIVLF
CCTCGGCGTGCCCAGCTTTCCTCTGCGGCGTGCACTACCTCTACTTCGCCATTGTGCTGTTCTTCTGCTCTGGCCTCCTCACCCTCACGGTCTCCCTGTGCACCGCGCCCATCCCCAGAA 1580 1600
c
- . M
K
E
-
D
V
1660
580 E
I
590 -
-
-
-
N
I
E
P
K
T
I
E
E
K
GSSLPVQNGCPESAM
HSKEEREDLDADEQQ 1740
1760
R
-
1780
620
610
600 F
1640
570 N
w
AGCACCTCCACCGCCTGGTCTTCAGTCTCCGGCATAGCAAGGAGGAACGGGAGGACCTGGATGCTGATGAGCAGCAAGGCTCCTCACTCCCTGTACAGAATGGGTGCCCAGAGAGTGCCA 1700 1720
IETQVPEKKKGI
L
1620
560 Y HLHRLVFSLR
550
FITIVVI
A
Y
D
630
LEQ---HGA
L
K
M
E
GGVGSPPPLTQEEAA
NFPQAPAPSLFRQCLLWFCGMSR
TGGAGATGAATGAGCCCCAGGCCCCGGCACCAAGCCTCTTCCGCCAGTGCCTGCTCTGGTTTTGTGGAATGAGCAGAGGTGGGGTGGGCAGTCCTCCGCCCCTTACCCAGGAGGAGGCAG 1840 1820
1860
1880
1900
12 /"
640 MKMKMT AAARRLEDI
67d
660
'650
T
t
SE~P~W~~V:N~N~::~~:VAVF~~~~~A*
CGGCAGCAGCCAGGCGGCTGGAGGACATCAGCGAGGACCCGAGCTGGGCCCGTGTGGTCAACCTCAAT~GCCCTGCTCATGATGGCAGTGGCCGTGTTCCTCTGGGGCTTCTATGCCTAAG 1940 1960
1980
2000
2020
ACCAACTGCGTTGGACACCATAAGCCACAGCCTCACAGGAAGTGGGGGTGAGGAGCCTGCGGTGCTCCCCAGAAAAGGGGAAGGGGCAGTGGGGTGAGAAGGTCCTGGCTCCCCTTCTCC 2080 2080
2100
2120
2140
CGGCCTTCCTCTGCCTGGGGCCCACTGCATCTGATTGGCAGTCACTTCCCATGAGGGCCTGGCCCACCCGCTGCAGTTGcccTAAGGAAAAATAAAGCTGCCTTTCCCCTGTAn 2200 2200
2220
2240
2260
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HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
cDNA
F461
Hu14 1
23
I
I
100
4
I
5
I
6
7
I
200
8
I
9
1
3Ao
1010all
12
I
400
I
I
I
5&
(Residue) SGLTl 1
23
4
5
6
7
7a
8
9 9a
10
11
I
I
500
Fig. 2. Hydrophobicity plots of Hu14 and SGLTI, with numbering of predicted membrane-spanning regions. Numbers of regions for SGLTI correspond to those in the original model (11). Regions 9a in SGLTI and IOa in Hul4 are predicted to be membrane surface rather than membrane-spanning regions.
600
(Residue) described (5). Hydrophobicity plots were determined by the Kyte-Doolittle method (17). Membrane-spanning regions of proteins were predicted by the method of Eisenberg et al. (6). RNA from human kidney cortex, human ileum, rabbit jejunum, various rat tissues (Sprague-Dawley rats), and the OK and LLC-PK, cell lines was prepared by the guanidinium isothiocyanate method using cesium-trifluoroacetic acid (TFA) as described by the manufacturer (Pharmacia). Poly(A)+ RNA was selected by oligo(dT) -cellulose column chromatography (Collaborative Research) and used for Northern blot analysis. RNA (2.5 pg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and was transferred to nitrocellulose filters. Blots were probed with 32P-labeled Hu14 and SGLTl cDNA inserts (37”C, 50% formamide) and washed in 0.1% SSC-0.1% SDS at 50°C (Figs. 4 and 5, right) and 53°C (Fig. 5, left). Probes (full-length cDNA inserts) were labeled using the T7QuickP rime system (Pharmacia). RNA ladder from GIBCO BRL Life Technologies was used as a size standard. cRNA was synthesized from the various pBluescript subclones in the presence of capping analogue as described previously (9). Every batch of cRNA was evaluated on formaldehyde gels to ensure its quality. Hu14 cRNA (35 ng) was used for in vitro translation in a rabbit reticulocyte lysate system (Promega) with L- [35S]methionine (New England Nuclear) for 1 h at 30°C as described earlier (10). Canine pancreatic microsomes (Promega) were added to some of the translation reactions, which were then centrifuged at 4°C for 30 min in buffer containing 2.5% glycerol. SDS sample buffer [125 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris HCl), pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 8 M urea, 20% sucrose, 0.5 mg/ml bromophenol blue] was added to all of the translation reactions, which were boiled for 5 min before SDS-polyacrylamide gel electrophoresis (PAGE) analysis using a 10% acrylamide gel. For functional analysis, in vitro transcribed Hu14 cRNA (30 ng/oocyte) were injected into collagenase-treated and manually defolliculated Xenopus oocytes. The uptake of 14C- or 3H-labeled substrates into injected oocytes over 1 h was measured in the presence of 100 mM Na+ 3 days after injection, as described l
(9). Each uptake is presented as the mean t SE of eight oocytes. Control oocytes were injected with water. All substrates were 14C-labeled except uridine, which was 3H-labeled. Concentrations were (in PM) 500 a-MeGlc; 50 galactose; 50 proline; and 100 uridine. The positive control for uptake experiments was rat SGLTl (R. Wells and M. Hediger, unpublished sequence) cRNA, which was synthesized and injected in parallel. Rat SGLTl-injected oocytes were assayed for the uptake of 500 PM 14C-labeled cu-MeGlc. Injected oocytes were also analyzed electrophysiologically. Oocytes were impaled with a single electrode, and the resting membrane potential was recorded in uptake medium (100 mM Na+) containing various sugars, amino acids, and vitamins (3-10 mM). The ability of these substrates to change the membrane potential was measured assuming that Na+-coupled cotransport would result in a membrane depolarization (25). Any changes were compared with those of water-injected control oocytes. RESULTS
AND
DISCUSSION
Low-stringency screening of human kidney library 1 using rabbit SGLTl as a probe resulted in the isolation of two clones that contained inserts larger than 1.5 kb: K7 (1.8 kb) and Kl5 (2.5 kb). Based on DNA sequencing and restriction mapping, K7 is a partial version of Kl5 lacking 0.6 kb at the Y-end. K15 was sequenced completely and has 69% DNA sequence identity to human SGLTl. It has an unusual (dA-dC)87 repeat at the V-end that is probably a cloning artifact. Similar repeats have previously been detected in tandem repeated genomic DNA (26) cDNA library 2 was screened using K15 as a probe. Clones with the following inserts were isolated: Hu5 (2.4 kb), HulO (2.6 kb), and Hu14 (2.4 kb). Their 3’- and V-ends and an internal region of Hu14 were sequenced. Hul4 was found to be identical to K15 except that it lacks the (dA-dC)87 repeat and has instead an additional 1’7
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F462
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
ALIDNPADIL ALIDNPADIA ELIRNAADIS RAVLETADIA . . . . MAISTP ..MQLEVILP
K15/Hu14 MEEHTEAGSA PEMG...AQK SNSTl MEEHMEAGSR LGLG...DHG SGLTl MDSSTWSPKT TAVTRPVETH SMIT . . . . . . . . . . . . . . . . . ..M PutP . . . . . . . . . . . . . . . . . . . . PanF . . . . . . . . . . . . . . . . . . . .
MLVTFCVYIF
cDNA
GMILIGFIAW GISVY#jRKR
LAM'@j+jTa
2
GSG GSG GSG GSE SGW SAS
TGAA TGAA TGAA SGAA AVFL AAYK
.. . .
AGFEWN WLL LFAPVYLTAG AGFEWN WLL LFAPVYLTAG LFVPIYIKAG GGFEWN LVVV GAWEFN LLQL VFIPIYIRSG GLTLGA WINWKLVA&jR LRVHTEYNNN IQLPAV MLGILGKK FAILAR.RYN A$jLNDmA
.. . . .. . . . SE; w ,GWV L
YLS YLS YLS YFA 11s WLA
IFTKISVDMF IFTKISVDMF IFTKISADIF IFTKLSVDLY FTIYCASGIV FVGAMTVQFI
DKYLGAATSL DKYMGAMTSL EKYMKAIPTI RRYMLASPNV EVI....... QTL.......
TVSEDP.AVG TVSEDP.AVG VSDGNT.TFQ TSILLTYNLS
4
NIYASVIA NIYASVIA NLYLAIFL NLYVSVIL SYETALWA PYETGLLI
1 QQAL
1 QQAL
INLAL IQESL FESTF LETAA
LG GACILMGYAF IA GAFILTGYAF LV GSLILTGFAF IV GALTLMIISM IF ALILTPVIVI HHARACDADW HRC.AAYWRS
WAPAAER
SGLF SGLF DAFM EEVK GDSL SNAV
6
NISSFCYRPR NISSSCYRPR EK.. .CYTPR NTNSCNVHPK . . . . . . . . .K . . . . . . . . .Q
PDSYHLLRHP PDSYHLLRDP ADSFHIFRDP KDALKMLRNP QKSIENVDML TIDPQLVTPQ
VT VT LT TD KG GAD@IL@F
LL LL FI FV SL
TIVSGW TIVSGW SILTLW TPASVW WGLGYF MTSFWVLVCF
YWCSDQVI YWCSDQVI YWCTDQVI YWCADQVI .. .. .. .. . . . . . . ..G.
HAR RISMTWMILC *VHRGIII
L GTIWAILMF
8 PDEVAC PDEVAC TEKIAC ADDIAC GIAYFN GMHL..
GTEV GTEV GTKV GSRA
GCSNIA GCSNIA GCTNIA GCSNIA
RAGD LLVG RAGE LLVGRLW RASE MIAGRLF SASS MIVGRIF FLRKH.A SQKELV...W I@PDQMQ *RLK...R
P.DL HVPDLV 10
9
WLPWQAAQG WLPWQAAQG WVPIVQSAQS WVPIIVEMQG LVAIALAANP ALLLLAAWKP
10a
GQLFDYIQAV GQLFDYIQSV GQLFDYIQSI GQMYLYIQEV ENRVLGLVSY PEMIIWLNLL
PPVS S.S S.S PPVS T.S PPIA A.D PPVA AWA AAFG AFG&AVFL
AVFVLALFVP AVFWALFVP AVFLLAIFWK ALFLLAIFWK PWLFSVMWS WPLVLGLYWE
LLTLT T LLIII T T ITIW LITVI T IGIW G LAFLVGNRFG
APIPRKHLHR APIPRKHLHR KPIPDVHLYR PPPTKEQIRT KAPSAAMQKR TSVPQATVLT
LVFSLRHS.. LVFSLRHS.. LCWSLRNS.. TTFWSKKSLV FAEADAHYHS TDK.......
LMGLA LMGLA LIGIS VLGAV L..TV VLYAV
RLIP RLIP RMIT RLTL IVWK LATL
SFGS SFGT YGT YRA WLG YLG
GSCVQPSACP GSCVRPSACP GSCMEPSNCP PECDQPDNRP . .. .. . .. .. . .. .. .. . ..
11
CSG CSG ISF VTG FGS .. . ... .. . . . . . . . . FH PIVPSLLLSL
.m........ .. .. .. .. . . e......... VKESCSPKDE APPSRLQES. .. .. . ... . .
. . .KEEREDL . . .KEEREDL . ..KEERIDL PYKMQEKSIL .. .. . .. .. . .. .. . .. .. .
DADEQQG.SS DADELEAPAS DAEEEN.... RCSENSEATN .. . .. .. . .. .. . .. .. . ..
LPVQNGCPES PPVQNGRPEH ..IQEGPKET HIIPNGKSED . .. . .. .. .. . .. . .. .. ..
.PAPSL AMEMNEPQA. ............................................ AVEMEEPQA. ............................................ .PGPGL IEIETQVPE. ........................................... ..KKKG I SIKGLQPEDV NLLVTCREEG NPVASLGHSE AETPVDAYSN GQAALMGEKE RKKETEDGGR
FRQCLLWFCG FRQCLLWFCG FRRAYDLFCG YWKFIDWFCG
MSRGGVGSPP MNRGRAGGPA LEQ...HGA P FKSKSLSKRS
PLTQEEAAAA PPTQEEEAAA KMTEEEEKAM LRDLMEEEAV
12
ARRLEDISED ARRLEDINED KMKMTDTSEK CLQM..LEEP
PSWARWNLN PRWSRWNLN PLWRTVLNVN PQVKLILNIG
ALLMMAVAVF ALLMMAVAMF GIILVTVAVF LFAVCSLGIF
LWGFYA. FWGFYA. 'CHAYFA. MFVYFSL
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
kb 7.5 -
4.4 -
2.4 -
1.35 -
1
2
3
Fig. 4. Northern blot analysis of human kidney cortex, human ileum, and rat jejunum mRNA. Blot was probed with labeled K15. Washing temperature was 50°C.
nucleotides at the Y-end. Hu5 appears to be identical to Hu14 except that it lacks 20 nucleotides at the Y-end. HulO has 5’- and 3’-sequences that are unrelated to Hu14. cRNA prepared from this clone did not translate in vitro using rabbit reticulocyte lysates (not shown). Hu14 was used for all further studies. The combined K15-Hu14 cDNA sequence of 2,271 nucleotides is shown in Fig. 1. There is a polyadenylation sequence (AATAAA) at position 2,251. There is one large open reading frame with an AUG translation start codon at position 21. This open reading frame (21-2,036) encodes a 672-residue protein, 10 residues longer than SGLTl. There are two potential NHs-linked glycosylation sites (Asn-250 and Asn-399). Residues that are different between SGLTl and K15Hu14 are indicated above the amino acid sequence in Fig.
cDNA
F463
1. There is 59% amino acid sequence identity between K15-Hu14 and human SGLTl, in contrast to the 84% identity between the human and rabbit SGLTl sequences (ll), suggesting that K15-Hu14 encodes a protein with different functional properties. K15-Hu14 has the same number of predicted membrane-spanning regions and hydrophobic and hydrophilic regions as SGLTl (ll), and they are in the same relative locations (Fig. 2), consistent with the hypothesis that the K15-Hu14 protein is a transporter with a secondary structure similar to that of SGLTl. At the amino acid sequence level, K15-Hu14 shows significant identity to the E. coli Na+ cotransporters of proline (p&P, 24%) (19) and pantothenic acid (panF, 25%) (14), the rabbit uridine-Na+ cotransporter (SNSTl, 91%) (22), and the canine myo-inositol-Na+ cotransporter (SMIT, 48%) (16). Several regions of similarity between human SGLTl and p&P have been noted previously (11). The similarity between K15-Hu14 and the recently described rabbit kidney Na+-nucleoside transporter SNSTl is striking and is found in all regions of the sequence except the 5’-end. As noted below, however, we find significant differences between the sites of expression and the uptake characteristics of these two clones. Figure 3 shows an alignment between the amino acid sequences of K15-Hu14, SGLTl, SNSTl, SMIT, p&P, and panF. Identical residues are found throughout the sequences. This high level of identity is suggestive of a common ancestry for procaryotic and eukaryotic Na+coupled transporters. The COOH-terminal region of SGLTl (aligned with residues 567-604 in Fig. 1) has distinct clusters of charged residues that may be involved in sugar binding through the formation of hydrogen bonds (9). Consistent with this hypothesis, the p&P and panF transporters are missing this region. The K15-Hu14 amino acid sequence has multiple charged residues in this area, although it is where the sequences of SGLTl and Hu14 differ most. If this is the ligand-binding part of the protein, it suggests that Hu14 encodes a transporter with a different substrate specificity or perhaps different binding affinity than SGLTl. The tissue distribution of Hu14 was evaluated by Northern blotting. Figure 4 shows the results of probing mRNA from human kidney cortex and ileum and rabbit jejunum. Strong hybridization to species at 2.4, 3.0, 3.5, and 4.5 kb is seen for kidney (lane I), and only weak hybridization is seen for human ileum (lane 2,3.0 kb) and rabbit jejunum (lane 3, 2.4 kb). Figure 5 shows different patterns of hybridization of human SGLTl (Fig. 5, left) and Hu14 (Fig. 5, right) probes to mRNA derived from various rat tissues and from OK and LLC-PKi cells. Hybridization to species at 4 and 2.4 kb in kidney and LLCPK, mRNA and at 4 kb in intestine are seen with the SGLTl probe. In contrast, the major hybridization seen with the Hu14 probe is to a 2.4-kb species in kidney and, to a lesser degree, intestine, OK, and LLC-PKi cell mRNA. Minor bands of 4-5 kb (probably representing SGLTl) are seen in kidney medulla, jejunum, and ileum.
Fig. 3. Alignment of amino acid sequences of Hu14, SGLTl, p&P, panF, SNSTl, and SMIT. Regions of 5 or 6 identical amino acids are boxed. Predicted membrane-spanning regions of K15-Hu14 (per Fig. 1) are shown by bold lines above sequences. * Location of Asp-to-Asn mutation in glucose-galactose malabsorption (23). Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
F464
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
cDNA
9.5 7.5
4.2 -
2.4 -
2.4
Human SGLTl
probe
Human Clone Hu 14 probe
Fig. 5. Northern blot analysis of rat tissues probed at 37°C with SGLTl (left) and Hu14 (right). Washing temperatures were 53 and 50°C for left and right panels, respectively. Same blot was probed with SGLTI, then stripped and reprobed with Hu14.
kDa
1 2
3 4
u-Me
Microsomes Endo H
-
-
+ + - +
Fig. 6. Autoradiograph of an SDS gel showing in vitro translation products of Hu14 cRNA. Reaction shown in lane 1 was performed in the absence of RNA; Hu14 cRNA was present for reactions shown in lanes 2-4. Addition of pancreatic microsomes and treatment with endoglycosidase H occurred as indicated.
This suggests that Hu14 is primarily expressed in the kidney, while SGLTl is expressed in the small intestine and to a lesser extent in the kidney. Under the hybridization conditions used, however, the possibility exists that hybridization occurred to an as yet uncharacterized RNA species with similarities to Hu14 and SGLTl (a reduced hybridization stringency was necessary to ensure hybridization of the human probe to rat mRNA). There is no hybridization signal seen for Hu14 in heart, a major site of expression of SNSTl (22). A rabbit reticulocyte lysate in vitro translation system was used to study whether the K15-Hu14 protein can be
Glucose
Galactose RADIOLABELED
PVJhe
Uridme
SUBSTRATE
Fig. 7. Uptake of radiolabeled substrates into Hu14 cRNA-injected oocytes. Control oocytes were injected with water. All substrates were 14C-labeled, except uridine, which was 3H-labeled. Concentrations were (in PM) 500 or-MeGlc; 50 galactose; 50 proline; and 100 uridine.
incorporated into pancreatic microsomes and posttranslationally processed (Fig. 6). With SDS-PAGE, we obtained a signal at 54 kDa in the absence and at 58 kDa in the presence of microsomes. Endoglycosidase H treatment converted the product obtained in the presence of microsomes back to 54 kDa, demonstrating that Hu14, like SGLTl (lo), is glycosylated. In an attempt to determine the function of Hu14, we injected Xenopus oocytes with Hu14 cRNA. Three days after injection, we measured the uptake of 14C-labeled cu-MeGlc, D-g&XtOSe, L-proline, and 3H-labeled uridine (Fig. 7). There was a slight increase in transport activity with these substrates (Fig. 7) and with D-&COSe and L-phenylalanine (not shown). The highest increase in uptake was obtained with 14C-labeled cy-MeGlc (500 PM)
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
(2-fold). Although statistically significant (P c O.OOOl), it is much less than the 189-fold increase in a-MeGlc uptake obtained with rat SGLTl cRNA-injected control oocytes (data not shown). There is no evidence of significant uridine transport (Fig. 7), despite a high degree of amino acid sequence identity (91%) between the Kl5-Hu14 protein and the Na+-uridine cotransporter SNSTl (22). In addition, we saw no evidence for Hu14-induced transport after testing more than 50 different substrates (hexoses, pentoses, amino acids, nucleosides, and vitamins, at l-10 mM) electrophysiologically in the presence of 100 mM Na+ for the ability to depolarize the resting membrane potential of Hu14 cRNA-injected oocytes. There are several possible explanations for the low level of Hul4-induced uptake. 1) Hu14 may encode a transporter for a substrate not yet tested, and the slight increases induced by Hu14 cRNA would be nonspecific and related to the high degree of structural similarity among the members of the Na+ cotransporter family (i.e., a “leakage” phenomenon). 2) Hu14 is incomplete at the 5’-end, although this seems unlikely given that the length of the Hu14 cRNA transcript corresponds in size to the major transcript on Northern blots (see Figs. 4 and 5), that the AUG initiation codon at position 21 has similarity to the Kozak consensus initiation sequence (15), and that the AUG initiation codon corresponds to the translation initiation sites of SGLTl and SNSTl. 3) The protein may not be properly translated, processed, or incorporated into the membranes of cRNA-injected Xenopus oocytes, although it is translated in an in vitro rabbit reticulocyte lysate translation mixture. 4) The Hu14 protein requires a cofactor or additional subunits. 5) Finally, the Hu14 protein may represent a low-affinity Na+-glucase cotransporter, although the level of uptake over controls is low. In summary, we have cloned a cDNA that appears to encode a new member of the Na+ cotransporter family, which includes SGLTl, SNSTl, SMIT, putP, and panF. Work is ongoing to characterize the function of this protein. We thank Drs. Steven Hebert and Kevin Ho for assistance with the electrophysiology experiments, Drs. Joseph Handler and Mo-Kwon for providing the SMIT sequence, Dr. David Rhoads for critical comments on the manuscript, and Paula Boutin for technical assistance. R. G. Wells is a Howard Hughes Medical Institute Research Fellow. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43632 and DK-43171 to M. A. Hediger and DK-19567 to E. M. Wright. Address for reprint requests: M. A. Hediger, Renal Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Received
2 January
1992; accepted
in final
form
1 April
1992.
REFERENCES
1. Barfuss, passive Physiol.
D. W., and J. Schafer. glucose transport 240 (Renal Fluid
Differences in active and along the proximal nephron. Am. J. Electrolyte Physiol. 9): F322-F332,1981.
2. Bell, G. I., N. M. Fong, M. M. Stempien, M. A. Wormstead, D. Caput, L. L. Ku, M. S. Urdea, L. B. Rall, and R. Sanchez-Pescador. Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. cleic Acids Res. 14: 8427-8446, 1986.
3. Coady,
M. J., A. M. Pajor,
and E. M. Wright. Sequence intestinal and renal Na+-glucose cotransport259 (Cell Physiol. 28): C605C610, 1990. Y., I. Yamato, and Y. Anraku. Nucleotide sequence the Na+/glutamate symport carrier gene of Escherichia
homologies among ers. Am. J. Physiol. 4.
Deguchi, of gltS,
Nu-
coli. Br. J. Biol.
Devereux,
Chem.
265: 21704-21708,
J., P. Haeberli,
sive set of sequence analysis Res. 12: 387-391, 1984.
Eisenberg,
F465
cDNA 1990.
and 0. Smithies. programs
D., E. Schwartz,
for the VAX.
M. Komaromy,
A comprehenNucleic Acids
and R. Wall.
Analysis of membrane hydrophobic moment
and surface protein sequences with the plot. J. MoZ. Biol. 179: 125-142, 1984. Elsas, L. J., and L. E. Rosenberg. Familial renal glycosuria: a genetic reappraisal of hexose transport by kidney and intestine. J. Clin. Invest. 48: 1845-1854, 1969.
Guastella, J., N. Nelson, H. Nelson, L. Czyzyk, S. Keynan, M. C. Miedel, N. Davidson, H. Lester, and B. Kanner. Cloning and expression of a rat brain Wash. DC 249: 1303-1306, 1990. 9.
transporter.
Hediger, M. A., M. J. Coady, T. S. Ikeda, Wright. Expression cloning and cDNA sequencing glucose
10.
GABA
Hediger,
co-transporter.
Nature
M. A., J. Mendlein,
Lond.
330: 379-381,
Science
and E. M. of the Na+/ 1987.
H.-S. Lee, and E. M. Wright.
Biosynthesis of the cloned intestinal Biochim. Biophys. Acta 1064: 360-364,
Na+/glucose 1991.
transporter.
M. A., E. Turk, and E. M. Wright. Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters. Proc. NatZ. Acad. Sci. USA 86: 5748-5752, 1989. 12. Hoffman, B. J., E. Mezey, and M. J. Brownstein. Cloning of a serotonin receptor affected by antidepressants. Science Wash. DC 254: 579-580, 1991. 13. Ikeda, T. S., E. S. Hwang, M. J. Coady, B. A. Hirayama, M. A. Hediger, and E. M. Wright. Characterization of a Na+/ glucose cotransporter cloned from rabbit small intestine. J. Membr. BioZ. 110: 87-95, 1989. 14. Jackowski, S., and J. H. Alix. Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli. J. Bacterial. 172: 3842-3848, 1990. 15. Kozak, M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196: 947-950, 1987. 16. Kwon, H. M., A. Yamauchi, S. Uchida, A. S. Preston, A. Garcia-Perez, M. Burg, and J. S. Handler. Cloning of the cDNA for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J. Biol. Chem. 267: 6297-6301, 1992. 17. Kyte, J., and R. F. Doolittle. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132, 1982. 18. Morrison, A. M., M. Panayotova-Heiermann, G. Feigl, B. Schoelermann, and R. K. H. Kinne. Sequence comparison of the sodium-D-glucose cotransport system in rabbit renal and intestinal epithelia. Biochim. Biophys. Acta 1089: 121-123, 1991. 19. Nakao, T., I. Yamato, and Y. Anraku. Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12. A!oI. Gen. Genet. 208: 70-75, 1987. 20. Ohta, T., K. J. Isselbacher, and D. B. Rhoads. Regulation of glucose transporters and LLC-PK, cells: effects of D-glucose and monosaccharides. Mol. CeZZ. BioZ. 10: 6941-6499, 1990. 21. Pacholczyk, T., R. D. Blakely, and S. Amara. Expression cloning of a cocaineand antidepressant-sensitive human noradrenaline transporter. Nature Lond. 350: 350-354, 1991. 22. Pajor, A. M., and E. M. Wright. Cloning and functional expression of a mammalian Na+/nucleoside cotransporter. J. BioZ. Chem. 267: 3557-3560, 1992. 23. Turk, E., B. Zabel, S. Mundlos, J. Dyer, and E. M. Wright. Glucose/galactose malabsorption caused by a defect in the Na+/ glucose cotransporter. Nature Lond. 28: 350, 354-356, 1991. 24. Turner, R. J., and A. Moran. Further studies of proximal tubular brush border membrane D-glucose transport heterogeneity. J. Membr. Biol. 70: 37-45, 1982. 25. Umbach, J. A., M. J. Coady, and E. M. Wright. Intestinal Na+/glucose transporter expressed in Xenopus oocytes is electrogenie. Biophys. J. 57: 1217-1224, 1990. 26. Weber, J. L., and P. E. May. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Human Genet. 44: 388-396, 1989. 27. Werner, A., M. L. Moore, H. Mantei, J. Biber, G. Semenza, and H. Murer. Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc. NcztZ. Acad. Sci. USA 88: 9608-9612, 1991. 11.
Hediger,
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
ANA M. PAJOR, AND MATTHIAS
YOSHIKATSU A. HEDIGER
KAN.AI,
ERIC
TURK,
Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Department of Physiology, School of Medicine, University of California, Los Angeles, California 90024 Wells, Rebecca G., Ana M. Pajor, Yoshikatsu Kanai, Eric Turk, Ernest M. Wright, and Matthias A. Hediger. Cloning of a human kidney cDNA with similarity to the sodium-glucosecotransporter. Am. J. Physiol. 263 (Renal Fhid Electrolyte Physiol. 32): F459-F465, 1992.-We have usedlowstringency screening with the human intestinal Na+-glucose cotransporter SGLTl to isolate a 2,271-nucleotide cDNA (Hul4) from human kidney. This clone, which encodesa 672residue protein, is 59% identical at the amino acid level to SGLTl and has a similar number and arrangementof predicted membrane-spanningregions.It alsosharessignificant sequence identity with other Na+-coupled transporters. Northern blot analysissuggestsstrong expressionof Hu14 in kidney, but, unlike SGLTl, no significant expression in intestine. We have been unable to demonstrate definitive transport of any of a number of substrates (including amino acids, sugars, nucleosides, and vitamins) into Hu14 cRNA-injected Xenopus oocytes, although sequenceconservation makesit likely that Hul4 representsanother member of the Na+ cotransporter family, possibly a secondNa+-glucosecotransporter. glucosetransport; small intestine
and kidney proximal tubule, glucose transport across epithelial cells proceeds via Na+-glucose cotransporters in the brush-border membrane and facilitated glucose transporters in the basolateral membrane. A brush-border Na+-glucose cotransporter (SGLTl) was previously isolated from rabbit (9) and human (11) small intestine, the LLC-PK1 renal cell line (20)) and rabbit (3, 18) and rat kidney cortex (R. Wells, unpublished results). A mutation at position 28 from Asp to Asn results in the human inherited disease glucose-galactose malabsorption (23). Efficient substrate transport in the kidney is provided by low- and high-affinity transporters arranged in series along the tubule. SGLTl from rabbit has the kinetic characteristics of a high-affinity transporter with a K, for methyl-cu-D-glucopyranoside (cu-MeGlc) of 110 PM and a Na+-to-glucose coupling ratio of 2:1 (13). SGLTl is probably the only Na+-glucose cotransporter expressed in the small intestine (13). There is evidence, however, from the studies of Turner and Moran (24) using rabbit renal proximal tubule brush-border membranes and from Barfuss and Schafer (1) using perfused proximal tubule segments for a second, low-affinity Na+-glucose cotransporter in kidney outer cortex with a Na+-to-glucose ratio of 1:l. The existence of a second Na+-glucose cotransporter in human kidney is supported by the observation of Elsas and Rosenberg (7) that renal but not intestinal glucose absorption is impaired in renal glycosuria, an autosomal recessive inborn error of transport. Several recently cloned Na+ cotransporters show significant similarity to SGLTI and suggest that there is a
IN THE SMALL
INTESTINE
0363-6127/92
$2.00
Copyright
family of such transporters: the Escherichia coli Na+proline (putP; see Ref. 19) and Na+-pantothenic acid (panF; see Ref. 14) transporters, the rabbit Na+-uridine cotransporter SNSTl (22), and the Na+-myo-inositol cotransporter (SMITl; see Ref. 16). The eukaryotic y-aminobutyric acid (8), serotonin (l2), and norepinephrine (21) Na+- and Cl--dependent transporters and the E. coli glutamate (4) and rabbit kidney phosphate (27) Na+-dependent transporters have recently been cloned, but have no significant sequence similarity to SGLTl. At present, there is no information available about the molecular basis of intestinal or renal Na+-coupled organic solute transporters other than SGLTl (9), SNSTl (22) and SMITl (16). In the present paper, cDNA libraries prepared from human kidney were screened at low stringency with a rabbit SGLTl probe to search for additional Na+ cotransporter-like sequences. METHODS
Cloneswere isolatedfrom the following human kidney cortex cDNA libraries: Library 1 (nonamplified, in X-Zap) prepared in our laboratories using human kidney tissue provided by Jill Norman, UCLA Schoolof Medicine, and library 2 (amplified, in A-gtl0) obtained from Graeme Bell, University of Chicago (2). Approximately 300,000cloneswere screenedfrom each library. Library 1 wasscreenedat 37°C with the gel-purified, 32P-labeled (T7QuickPrime) rabbit SGLTI Eco RI insert (9). The hybridization solution contained 5~ standard sodium citrate (SSC), 3~ Denhardt’s solution, 0.2% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 50% formamide, 0.01% antifoam B (Sigma), 0.1 mg/ml yeast RNA (Sigma, phenol extracted), 0.2 mg/ml denatured calf thymus DNA (Lofstrand), 2.5 mM sodium pyrophosphate, and 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5. The filters were washedin 0.1~ SSC-0.1% SDS at 50°C. The following two cloneswith inserts larger than 1.5 kb were selectedfor further analysis:K7 (1.8 kb) and K15 (2.5 kb). Clone K15 was used as a probe to screen library 2. The screeningconditions were the sameasfor the first library except that the filters were washedat higher stringency (0.1X SSC-0.1% SDS at 65°C). Multiple clones were selected, including Hu5 (2.4 kb), Hull0 (2.6 kb), and Hu14’ (2.4 kb). Both strands of clone K15, the 5’- and 3’-ends of Hu5, HulO, and Hu14, and an internal region of Hu14 were sequenced.K15 was subclonedinto Ml3 mp19 and sequencedby the dideoxy-sequencing method using 35S-dATP. Isolated h-phagecDNA inserts from Hu5, HulO, and Hu14 were subclonedinto pBluescript (Stratagene) and used for double-stranding sequencing with a polymerase dideoxysequencingkit (Pharmacia). Synthetic oligonucleotideprimers were usedto complete sequencing. A computer-basedsearchof the Genbank, EMBL, Swissprot, and Pir databaseswasperformed, and sequences were alignedas l The GenBank
nucleotide sequence of clone Hu14 data base (accession no. M95549).
0 1992 the American
Physiological
Society
has been deposited
in the F459
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
1 1 SGLTl:
10
I
20
MDSSTWSPKTTAVTRPVETHE
R
30
s
A
v
I
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Hu14: MEEHTEAGSAPEMGAQKALIDNPADILVIAAYFL GGGGGCAGATCCTGGGGAGAATGGAGGAGCACACAGAGGCAGGCTCGGCACCAGAGATGGGGGCCCAGAAGGCCCTGATTGACAATCCTGCTGACATCCTAGTCATTGCTGCATATTTCC 20
40
40 V
M
60
1
A
A
80
50 F
100
60
s
70
F
I
LVIGVGLWSMCRTNRGTVGGYFLAGRSMVWWPVGASLFAS TGCTGGTCATTGGCGTTGGCTTGTGGTCCATGTGCAGAACCAACAGAGGCACTGTGGGCGGCTACTTCCTGGCAGGACGCAGCATGGTGTGGTGGCCGGTTGGGGCCTCTCTCTTCGCCA 140
160
180
200
I
220
G
v
L
v
v
V
NIGSGHFVGLAGTGAASGLAVAGFEWNALFVVLLLGWLFA GCAACATCGGCAGTGGCCACTTTGTGGGCCTGGCAGGGACTGGCGCTGCAAGTGGCTTGGCTGTTGCTGGATTCGAGTGGAATGCGCTCTTCGTGGTGCTGCTACTGGGCTGGCTGTTTG 260
280
120
I
I
300
I
K
320
130
V
340
140
E
Q
Q
150
v
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PVYLTAGVITMPQYLRKRFGGRRIRLYLSVLSLFLYIFTK CACCCGTGTACCTGACAGCGGGGGTCATCACGATGCCACAGTACCTGCGCAAGCGCTTCGGCGGCCGCCGCATCCGCCTCTACCTGTCTGTGCTCTCCCTTTTCCTGTACATCTTCACCA 380 400
420
160
h70
A I I N L ISVDMFSGAVFIQQALGWNIYASVIALLGITMIYTVTGGL AGATCTCAGTGGACATGTTCTCCGGAGCTGTATTCATCCAGCAGGCTCTGGGCTGGAACATCTATGCCTCCGTCATCGCGCTTCTGGGCATCACCATGATTTACACGGTGACAGGAGGGC 500
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4 180
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580
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AALMYTDTVQTFVILGGACILMGYAFHEVGGYSGLFDKYL TGGCCGCGCTGATGTACACGGACACGGTACAGACCTTCGTCATTCTGGGGGGCGCCTGCATCCTCATGGGTTACGCCTTCCACGAGGTGGGCGGGTATTCGGGTCTCTTCGACAAATACC 620
640
660.
240
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P
680
250
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-DGNTTFQEK
700
260 A
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GAATSLTVSEDPAVGNISSFCYRPRPDSYHLLRHPVTGDL TGGGAGCAGCGACTTCGCTGACGGTGTCCGAGGATCCAGCCGTGGGAAACATCTCCAGCTTCTGCTATCGACCCCGGCCCGACTCCTACCACCTGCTCCGGCACCCCGTGACCGGGGATC 740
760
780
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PWPALLLGLTIVSGWYWCSDQVIVQRCLAGKSLTHIKAGC TGCCGTGGCCCGCGCTGCTCCTCGGACTCACAATCGTCTCGGGCTGGTACTGGTGCAGCGACCAGGTC~TCGTGCAGCGCTGCCTGGCCGGGAAGAGCCTGACCCACATCAAGGCGGGCT 860
880
900
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7 320
330
7
340
350 T
ILCGYLKLTPMFLMVMPGMI
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KI
s
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SRILYPDEVACVVPEVCRRV
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1020
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T
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CGTEVGCSNI
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TGTGCGGCACGGAGGTGGGCTGCTCCAACATCGCCTACCCGCGGCTCGTCGTGAAGCTCATGCCCAACGGTCTGCGCGGACTCATGCTGGCGGTCATGCTGGCCGCGCTCATGTCCTCGC 1120 1100
1140
S
1160
Fig. 1. Nucleotide sequence and deduced amino acid sequence of Hu14-K15. Residues that are different in human SGLTI are shown above amino acid sequence. Brackets indicate predicted membrane-spanning regions. * First stop codon (TAA).
1180 9
40d
410
420 A
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TGGTAGTGTCGGTGGCCTGGCTTCCCGTGGTGCAGGCGGCACAGGGCGGGCAGCTCTTCGATTACATCCAGGCAGTCTCTAGCTACCTGGCACCGCCCGTGTCCGCCGTCTTCGTGCTGG 1340 1360
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LFVPRVNEQGAFWGLIGGLLMGLARLIPEFSFGSGSCVQP CGCTCTTCGTGCCGCGCGTTAATGAGCAGGGCGCCTTCTGGGGACTCATCGGGGGCCTGCTGATGGGCCTGGCACGCCTGATTCCCGAGTTCTCCTTCGGCTCGGGCAGCTGTGTGCAGC 1460 1480
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GGVGSPPPLTQEEAA
NFPQAPAPSLFRQCLLWFCGMSR
TGGAGATGAATGAGCCCCAGGCCCCGGCACCAAGCCTCTTCCGCCAGTGCCTGCTCTGGTTTTGTGGAATGAGCAGAGGTGGGGTGGGCAGTCCTCCGCCCCTTACCCAGGAGGAGGCAG 1840 1820
1860
1880
1900
12 /"
640 MKMKMT AAARRLEDI
67d
660
'650
T
t
SE~P~W~~V:N~N~::~~:VAVF~~~~~A*
CGGCAGCAGCCAGGCGGCTGGAGGACATCAGCGAGGACCCGAGCTGGGCCCGTGTGGTCAACCTCAAT~GCCCTGCTCATGATGGCAGTGGCCGTGTTCCTCTGGGGCTTCTATGCCTAAG 1940 1960
1980
2000
2020
ACCAACTGCGTTGGACACCATAAGCCACAGCCTCACAGGAAGTGGGGGTGAGGAGCCTGCGGTGCTCCCCAGAAAAGGGGAAGGGGCAGTGGGGTGAGAAGGTCCTGGCTCCCCTTCTCC 2080 2080
2100
2120
2140
CGGCCTTCCTCTGCCTGGGGCCCACTGCATCTGATTGGCAGTCACTTCCCATGAGGGCCTGGCCCACCCGCTGCAGTTGcccTAAGGAAAAATAAAGCTGCCTTTCCCCTGTAn 2200 2200
2220
2240
2260
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
cDNA
F461
Hu14 1
23
I
I
100
4
I
5
I
6
7
I
200
8
I
9
1
3Ao
1010all
12
I
400
I
I
I
5&
(Residue) SGLTl 1
23
4
5
6
7
7a
8
9 9a
10
11
I
I
500
Fig. 2. Hydrophobicity plots of Hu14 and SGLTI, with numbering of predicted membrane-spanning regions. Numbers of regions for SGLTI correspond to those in the original model (11). Regions 9a in SGLTI and IOa in Hul4 are predicted to be membrane surface rather than membrane-spanning regions.
600
(Residue) described (5). Hydrophobicity plots were determined by the Kyte-Doolittle method (17). Membrane-spanning regions of proteins were predicted by the method of Eisenberg et al. (6). RNA from human kidney cortex, human ileum, rabbit jejunum, various rat tissues (Sprague-Dawley rats), and the OK and LLC-PK, cell lines was prepared by the guanidinium isothiocyanate method using cesium-trifluoroacetic acid (TFA) as described by the manufacturer (Pharmacia). Poly(A)+ RNA was selected by oligo(dT) -cellulose column chromatography (Collaborative Research) and used for Northern blot analysis. RNA (2.5 pg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and was transferred to nitrocellulose filters. Blots were probed with 32P-labeled Hu14 and SGLTl cDNA inserts (37”C, 50% formamide) and washed in 0.1% SSC-0.1% SDS at 50°C (Figs. 4 and 5, right) and 53°C (Fig. 5, left). Probes (full-length cDNA inserts) were labeled using the T7QuickP rime system (Pharmacia). RNA ladder from GIBCO BRL Life Technologies was used as a size standard. cRNA was synthesized from the various pBluescript subclones in the presence of capping analogue as described previously (9). Every batch of cRNA was evaluated on formaldehyde gels to ensure its quality. Hu14 cRNA (35 ng) was used for in vitro translation in a rabbit reticulocyte lysate system (Promega) with L- [35S]methionine (New England Nuclear) for 1 h at 30°C as described earlier (10). Canine pancreatic microsomes (Promega) were added to some of the translation reactions, which were then centrifuged at 4°C for 30 min in buffer containing 2.5% glycerol. SDS sample buffer [125 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris HCl), pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 8 M urea, 20% sucrose, 0.5 mg/ml bromophenol blue] was added to all of the translation reactions, which were boiled for 5 min before SDS-polyacrylamide gel electrophoresis (PAGE) analysis using a 10% acrylamide gel. For functional analysis, in vitro transcribed Hu14 cRNA (30 ng/oocyte) were injected into collagenase-treated and manually defolliculated Xenopus oocytes. The uptake of 14C- or 3H-labeled substrates into injected oocytes over 1 h was measured in the presence of 100 mM Na+ 3 days after injection, as described l
(9). Each uptake is presented as the mean t SE of eight oocytes. Control oocytes were injected with water. All substrates were 14C-labeled except uridine, which was 3H-labeled. Concentrations were (in PM) 500 a-MeGlc; 50 galactose; 50 proline; and 100 uridine. The positive control for uptake experiments was rat SGLTl (R. Wells and M. Hediger, unpublished sequence) cRNA, which was synthesized and injected in parallel. Rat SGLTl-injected oocytes were assayed for the uptake of 500 PM 14C-labeled cu-MeGlc. Injected oocytes were also analyzed electrophysiologically. Oocytes were impaled with a single electrode, and the resting membrane potential was recorded in uptake medium (100 mM Na+) containing various sugars, amino acids, and vitamins (3-10 mM). The ability of these substrates to change the membrane potential was measured assuming that Na+-coupled cotransport would result in a membrane depolarization (25). Any changes were compared with those of water-injected control oocytes. RESULTS
AND
DISCUSSION
Low-stringency screening of human kidney library 1 using rabbit SGLTl as a probe resulted in the isolation of two clones that contained inserts larger than 1.5 kb: K7 (1.8 kb) and Kl5 (2.5 kb). Based on DNA sequencing and restriction mapping, K7 is a partial version of Kl5 lacking 0.6 kb at the Y-end. K15 was sequenced completely and has 69% DNA sequence identity to human SGLTl. It has an unusual (dA-dC)87 repeat at the V-end that is probably a cloning artifact. Similar repeats have previously been detected in tandem repeated genomic DNA (26) cDNA library 2 was screened using K15 as a probe. Clones with the following inserts were isolated: Hu5 (2.4 kb), HulO (2.6 kb), and Hu14 (2.4 kb). Their 3’- and V-ends and an internal region of Hu14 were sequenced. Hul4 was found to be identical to K15 except that it lacks the (dA-dC)87 repeat and has instead an additional 1’7
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F462
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
ALIDNPADIL ALIDNPADIA ELIRNAADIS RAVLETADIA . . . . MAISTP ..MQLEVILP
K15/Hu14 MEEHTEAGSA PEMG...AQK SNSTl MEEHMEAGSR LGLG...DHG SGLTl MDSSTWSPKT TAVTRPVETH SMIT . . . . . . . . . . . . . . . . . ..M PutP . . . . . . . . . . . . . . . . . . . . PanF . . . . . . . . . . . . . . . . . . . .
MLVTFCVYIF
cDNA
GMILIGFIAW GISVY#jRKR
LAM'@j+jTa
2
GSG GSG GSG GSE SGW SAS
TGAA TGAA TGAA SGAA AVFL AAYK
.. . .
AGFEWN WLL LFAPVYLTAG AGFEWN WLL LFAPVYLTAG LFVPIYIKAG GGFEWN LVVV GAWEFN LLQL VFIPIYIRSG GLTLGA WINWKLVA&jR LRVHTEYNNN IQLPAV MLGILGKK FAILAR.RYN A$jLNDmA
.. . . .. . . . SE; w ,GWV L
YLS YLS YLS YFA 11s WLA
IFTKISVDMF IFTKISVDMF IFTKISADIF IFTKLSVDLY FTIYCASGIV FVGAMTVQFI
DKYLGAATSL DKYMGAMTSL EKYMKAIPTI RRYMLASPNV EVI....... QTL.......
TVSEDP.AVG TVSEDP.AVG VSDGNT.TFQ TSILLTYNLS
4
NIYASVIA NIYASVIA NLYLAIFL NLYVSVIL SYETALWA PYETGLLI
1 QQAL
1 QQAL
INLAL IQESL FESTF LETAA
LG GACILMGYAF IA GAFILTGYAF LV GSLILTGFAF IV GALTLMIISM IF ALILTPVIVI HHARACDADW HRC.AAYWRS
WAPAAER
SGLF SGLF DAFM EEVK GDSL SNAV
6
NISSFCYRPR NISSSCYRPR EK.. .CYTPR NTNSCNVHPK . . . . . . . . .K . . . . . . . . .Q
PDSYHLLRHP PDSYHLLRDP ADSFHIFRDP KDALKMLRNP QKSIENVDML TIDPQLVTPQ
VT VT LT TD KG GAD@IL@F
LL LL FI FV SL
TIVSGW TIVSGW SILTLW TPASVW WGLGYF MTSFWVLVCF
YWCSDQVI YWCSDQVI YWCTDQVI YWCADQVI .. .. .. .. . . . . . . ..G.
HAR RISMTWMILC *VHRGIII
L GTIWAILMF
8 PDEVAC PDEVAC TEKIAC ADDIAC GIAYFN GMHL..
GTEV GTEV GTKV GSRA
GCSNIA GCSNIA GCTNIA GCSNIA
RAGD LLVG RAGE LLVGRLW RASE MIAGRLF SASS MIVGRIF FLRKH.A SQKELV...W I@PDQMQ *RLK...R
P.DL HVPDLV 10
9
WLPWQAAQG WLPWQAAQG WVPIVQSAQS WVPIIVEMQG LVAIALAANP ALLLLAAWKP
10a
GQLFDYIQAV GQLFDYIQSV GQLFDYIQSI GQMYLYIQEV ENRVLGLVSY PEMIIWLNLL
PPVS S.S S.S PPVS T.S PPIA A.D PPVA AWA AAFG AFG&AVFL
AVFVLALFVP AVFWALFVP AVFLLAIFWK ALFLLAIFWK PWLFSVMWS WPLVLGLYWE
LLTLT T LLIII T T ITIW LITVI T IGIW G LAFLVGNRFG
APIPRKHLHR APIPRKHLHR KPIPDVHLYR PPPTKEQIRT KAPSAAMQKR TSVPQATVLT
LVFSLRHS.. LVFSLRHS.. LCWSLRNS.. TTFWSKKSLV FAEADAHYHS TDK.......
LMGLA LMGLA LIGIS VLGAV L..TV VLYAV
RLIP RLIP RMIT RLTL IVWK LATL
SFGS SFGT YGT YRA WLG YLG
GSCVQPSACP GSCVRPSACP GSCMEPSNCP PECDQPDNRP . .. .. . .. .. . .. .. .. . ..
11
CSG CSG ISF VTG FGS .. . ... .. . . . . . . . . FH PIVPSLLLSL
.m........ .. .. .. .. . . e......... VKESCSPKDE APPSRLQES. .. .. . ... . .
. . .KEEREDL . . .KEEREDL . ..KEERIDL PYKMQEKSIL .. .. . .. .. . .. .. . .. .. .
DADEQQG.SS DADELEAPAS DAEEEN.... RCSENSEATN .. . .. .. . .. .. . .. .. . ..
LPVQNGCPES PPVQNGRPEH ..IQEGPKET HIIPNGKSED . .. . .. .. .. . .. . .. .. ..
.PAPSL AMEMNEPQA. ............................................ AVEMEEPQA. ............................................ .PGPGL IEIETQVPE. ........................................... ..KKKG I SIKGLQPEDV NLLVTCREEG NPVASLGHSE AETPVDAYSN GQAALMGEKE RKKETEDGGR
FRQCLLWFCG FRQCLLWFCG FRRAYDLFCG YWKFIDWFCG
MSRGGVGSPP MNRGRAGGPA LEQ...HGA P FKSKSLSKRS
PLTQEEAAAA PPTQEEEAAA KMTEEEEKAM LRDLMEEEAV
12
ARRLEDISED ARRLEDINED KMKMTDTSEK CLQM..LEEP
PSWARWNLN PRWSRWNLN PLWRTVLNVN PQVKLILNIG
ALLMMAVAVF ALLMMAVAMF GIILVTVAVF LFAVCSLGIF
LWGFYA. FWGFYA. 'CHAYFA. MFVYFSL
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HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
kb 7.5 -
4.4 -
2.4 -
1.35 -
1
2
3
Fig. 4. Northern blot analysis of human kidney cortex, human ileum, and rat jejunum mRNA. Blot was probed with labeled K15. Washing temperature was 50°C.
nucleotides at the Y-end. Hu5 appears to be identical to Hu14 except that it lacks 20 nucleotides at the Y-end. HulO has 5’- and 3’-sequences that are unrelated to Hu14. cRNA prepared from this clone did not translate in vitro using rabbit reticulocyte lysates (not shown). Hu14 was used for all further studies. The combined K15-Hu14 cDNA sequence of 2,271 nucleotides is shown in Fig. 1. There is a polyadenylation sequence (AATAAA) at position 2,251. There is one large open reading frame with an AUG translation start codon at position 21. This open reading frame (21-2,036) encodes a 672-residue protein, 10 residues longer than SGLTl. There are two potential NHs-linked glycosylation sites (Asn-250 and Asn-399). Residues that are different between SGLTl and K15Hu14 are indicated above the amino acid sequence in Fig.
cDNA
F463
1. There is 59% amino acid sequence identity between K15-Hu14 and human SGLTl, in contrast to the 84% identity between the human and rabbit SGLTl sequences (ll), suggesting that K15-Hu14 encodes a protein with different functional properties. K15-Hu14 has the same number of predicted membrane-spanning regions and hydrophobic and hydrophilic regions as SGLTl (ll), and they are in the same relative locations (Fig. 2), consistent with the hypothesis that the K15-Hu14 protein is a transporter with a secondary structure similar to that of SGLTl. At the amino acid sequence level, K15-Hu14 shows significant identity to the E. coli Na+ cotransporters of proline (p&P, 24%) (19) and pantothenic acid (panF, 25%) (14), the rabbit uridine-Na+ cotransporter (SNSTl, 91%) (22), and the canine myo-inositol-Na+ cotransporter (SMIT, 48%) (16). Several regions of similarity between human SGLTl and p&P have been noted previously (11). The similarity between K15-Hu14 and the recently described rabbit kidney Na+-nucleoside transporter SNSTl is striking and is found in all regions of the sequence except the 5’-end. As noted below, however, we find significant differences between the sites of expression and the uptake characteristics of these two clones. Figure 3 shows an alignment between the amino acid sequences of K15-Hu14, SGLTl, SNSTl, SMIT, p&P, and panF. Identical residues are found throughout the sequences. This high level of identity is suggestive of a common ancestry for procaryotic and eukaryotic Na+coupled transporters. The COOH-terminal region of SGLTl (aligned with residues 567-604 in Fig. 1) has distinct clusters of charged residues that may be involved in sugar binding through the formation of hydrogen bonds (9). Consistent with this hypothesis, the p&P and panF transporters are missing this region. The K15-Hu14 amino acid sequence has multiple charged residues in this area, although it is where the sequences of SGLTl and Hu14 differ most. If this is the ligand-binding part of the protein, it suggests that Hu14 encodes a transporter with a different substrate specificity or perhaps different binding affinity than SGLTl. The tissue distribution of Hu14 was evaluated by Northern blotting. Figure 4 shows the results of probing mRNA from human kidney cortex and ileum and rabbit jejunum. Strong hybridization to species at 2.4, 3.0, 3.5, and 4.5 kb is seen for kidney (lane I), and only weak hybridization is seen for human ileum (lane 2,3.0 kb) and rabbit jejunum (lane 3, 2.4 kb). Figure 5 shows different patterns of hybridization of human SGLTl (Fig. 5, left) and Hu14 (Fig. 5, right) probes to mRNA derived from various rat tissues and from OK and LLC-PKi cells. Hybridization to species at 4 and 2.4 kb in kidney and LLCPK, mRNA and at 4 kb in intestine are seen with the SGLTl probe. In contrast, the major hybridization seen with the Hu14 probe is to a 2.4-kb species in kidney and, to a lesser degree, intestine, OK, and LLC-PKi cell mRNA. Minor bands of 4-5 kb (probably representing SGLTl) are seen in kidney medulla, jejunum, and ileum.
Fig. 3. Alignment of amino acid sequences of Hu14, SGLTl, p&P, panF, SNSTl, and SMIT. Regions of 5 or 6 identical amino acids are boxed. Predicted membrane-spanning regions of K15-Hu14 (per Fig. 1) are shown by bold lines above sequences. * Location of Asp-to-Asn mutation in glucose-galactose malabsorption (23). Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 23, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
F464
HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
cDNA
9.5 7.5
4.2 -
2.4 -
2.4
Human SGLTl
probe
Human Clone Hu 14 probe
Fig. 5. Northern blot analysis of rat tissues probed at 37°C with SGLTl (left) and Hu14 (right). Washing temperatures were 53 and 50°C for left and right panels, respectively. Same blot was probed with SGLTI, then stripped and reprobed with Hu14.
kDa
1 2
3 4
u-Me
Microsomes Endo H
-
-
+ + - +
Fig. 6. Autoradiograph of an SDS gel showing in vitro translation products of Hu14 cRNA. Reaction shown in lane 1 was performed in the absence of RNA; Hu14 cRNA was present for reactions shown in lanes 2-4. Addition of pancreatic microsomes and treatment with endoglycosidase H occurred as indicated.
This suggests that Hu14 is primarily expressed in the kidney, while SGLTl is expressed in the small intestine and to a lesser extent in the kidney. Under the hybridization conditions used, however, the possibility exists that hybridization occurred to an as yet uncharacterized RNA species with similarities to Hu14 and SGLTl (a reduced hybridization stringency was necessary to ensure hybridization of the human probe to rat mRNA). There is no hybridization signal seen for Hu14 in heart, a major site of expression of SNSTl (22). A rabbit reticulocyte lysate in vitro translation system was used to study whether the K15-Hu14 protein can be
Glucose
Galactose RADIOLABELED
PVJhe
Uridme
SUBSTRATE
Fig. 7. Uptake of radiolabeled substrates into Hu14 cRNA-injected oocytes. Control oocytes were injected with water. All substrates were 14C-labeled, except uridine, which was 3H-labeled. Concentrations were (in PM) 500 or-MeGlc; 50 galactose; 50 proline; and 100 uridine.
incorporated into pancreatic microsomes and posttranslationally processed (Fig. 6). With SDS-PAGE, we obtained a signal at 54 kDa in the absence and at 58 kDa in the presence of microsomes. Endoglycosidase H treatment converted the product obtained in the presence of microsomes back to 54 kDa, demonstrating that Hu14, like SGLTl (lo), is glycosylated. In an attempt to determine the function of Hu14, we injected Xenopus oocytes with Hu14 cRNA. Three days after injection, we measured the uptake of 14C-labeled cu-MeGlc, D-g&XtOSe, L-proline, and 3H-labeled uridine (Fig. 7). There was a slight increase in transport activity with these substrates (Fig. 7) and with D-&COSe and L-phenylalanine (not shown). The highest increase in uptake was obtained with 14C-labeled cy-MeGlc (500 PM)
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HUMAN
NA+-GLUCOSE
TRANSPORTER-RELATED
(2-fold). Although statistically significant (P c O.OOOl), it is much less than the 189-fold increase in a-MeGlc uptake obtained with rat SGLTl cRNA-injected control oocytes (data not shown). There is no evidence of significant uridine transport (Fig. 7), despite a high degree of amino acid sequence identity (91%) between the Kl5-Hu14 protein and the Na+-uridine cotransporter SNSTl (22). In addition, we saw no evidence for Hu14-induced transport after testing more than 50 different substrates (hexoses, pentoses, amino acids, nucleosides, and vitamins, at l-10 mM) electrophysiologically in the presence of 100 mM Na+ for the ability to depolarize the resting membrane potential of Hu14 cRNA-injected oocytes. There are several possible explanations for the low level of Hul4-induced uptake. 1) Hu14 may encode a transporter for a substrate not yet tested, and the slight increases induced by Hu14 cRNA would be nonspecific and related to the high degree of structural similarity among the members of the Na+ cotransporter family (i.e., a “leakage” phenomenon). 2) Hu14 is incomplete at the 5’-end, although this seems unlikely given that the length of the Hu14 cRNA transcript corresponds in size to the major transcript on Northern blots (see Figs. 4 and 5), that the AUG initiation codon at position 21 has similarity to the Kozak consensus initiation sequence (15), and that the AUG initiation codon corresponds to the translation initiation sites of SGLTl and SNSTl. 3) The protein may not be properly translated, processed, or incorporated into the membranes of cRNA-injected Xenopus oocytes, although it is translated in an in vitro rabbit reticulocyte lysate translation mixture. 4) The Hu14 protein requires a cofactor or additional subunits. 5) Finally, the Hu14 protein may represent a low-affinity Na+-glucase cotransporter, although the level of uptake over controls is low. In summary, we have cloned a cDNA that appears to encode a new member of the Na+ cotransporter family, which includes SGLTl, SNSTl, SMIT, putP, and panF. Work is ongoing to characterize the function of this protein. We thank Drs. Steven Hebert and Kevin Ho for assistance with the electrophysiology experiments, Drs. Joseph Handler and Mo-Kwon for providing the SMIT sequence, Dr. David Rhoads for critical comments on the manuscript, and Paula Boutin for technical assistance. R. G. Wells is a Howard Hughes Medical Institute Research Fellow. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43632 and DK-43171 to M. A. Hediger and DK-19567 to E. M. Wright. Address for reprint requests: M. A. Hediger, Renal Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Received
2 January
1992; accepted
in final
form
1 April
1992.
REFERENCES
1. Barfuss, passive Physiol.
D. W., and J. Schafer. glucose transport 240 (Renal Fluid
Differences in active and along the proximal nephron. Am. J. Electrolyte Physiol. 9): F322-F332,1981.
2. Bell, G. I., N. M. Fong, M. M. Stempien, M. A. Wormstead, D. Caput, L. L. Ku, M. S. Urdea, L. B. Rall, and R. Sanchez-Pescador. Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. cleic Acids Res. 14: 8427-8446, 1986.
3. Coady,
M. J., A. M. Pajor,
and E. M. Wright. Sequence intestinal and renal Na+-glucose cotransport259 (Cell Physiol. 28): C605C610, 1990. Y., I. Yamato, and Y. Anraku. Nucleotide sequence the Na+/glutamate symport carrier gene of Escherichia
homologies among ers. Am. J. Physiol. 4.
Deguchi, of gltS,
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coli. Br. J. Biol.
Devereux,
Chem.
265: 21704-21708,
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sive set of sequence analysis Res. 12: 387-391, 1984.
Eisenberg,
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cDNA 1990.
and 0. Smithies. programs
D., E. Schwartz,
for the VAX.
M. Komaromy,
A comprehenNucleic Acids
and R. Wall.
Analysis of membrane hydrophobic moment
and surface protein sequences with the plot. J. MoZ. Biol. 179: 125-142, 1984. Elsas, L. J., and L. E. Rosenberg. Familial renal glycosuria: a genetic reappraisal of hexose transport by kidney and intestine. J. Clin. Invest. 48: 1845-1854, 1969.
Guastella, J., N. Nelson, H. Nelson, L. Czyzyk, S. Keynan, M. C. Miedel, N. Davidson, H. Lester, and B. Kanner. Cloning and expression of a rat brain Wash. DC 249: 1303-1306, 1990. 9.
transporter.
Hediger, M. A., M. J. Coady, T. S. Ikeda, Wright. Expression cloning and cDNA sequencing glucose
10.
GABA
Hediger,
co-transporter.
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M. A., J. Mendlein,
Lond.
330: 379-381,
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and E. M. of the Na+/ 1987.
H.-S. Lee, and E. M. Wright.
Biosynthesis of the cloned intestinal Biochim. Biophys. Acta 1064: 360-364,
Na+/glucose 1991.
transporter.
M. A., E. Turk, and E. M. Wright. Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters. Proc. NatZ. Acad. Sci. USA 86: 5748-5752, 1989. 12. Hoffman, B. J., E. Mezey, and M. J. Brownstein. Cloning of a serotonin receptor affected by antidepressants. Science Wash. DC 254: 579-580, 1991. 13. Ikeda, T. S., E. S. Hwang, M. J. Coady, B. A. Hirayama, M. A. Hediger, and E. M. Wright. Characterization of a Na+/ glucose cotransporter cloned from rabbit small intestine. J. Membr. BioZ. 110: 87-95, 1989. 14. Jackowski, S., and J. H. Alix. Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli. J. Bacterial. 172: 3842-3848, 1990. 15. Kozak, M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196: 947-950, 1987. 16. Kwon, H. M., A. Yamauchi, S. Uchida, A. S. Preston, A. Garcia-Perez, M. Burg, and J. S. Handler. Cloning of the cDNA for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J. Biol. Chem. 267: 6297-6301, 1992. 17. Kyte, J., and R. F. Doolittle. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132, 1982. 18. Morrison, A. M., M. Panayotova-Heiermann, G. Feigl, B. Schoelermann, and R. K. H. Kinne. Sequence comparison of the sodium-D-glucose cotransport system in rabbit renal and intestinal epithelia. Biochim. Biophys. Acta 1089: 121-123, 1991. 19. Nakao, T., I. Yamato, and Y. Anraku. Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12. A!oI. Gen. Genet. 208: 70-75, 1987. 20. Ohta, T., K. J. Isselbacher, and D. B. Rhoads. Regulation of glucose transporters and LLC-PK, cells: effects of D-glucose and monosaccharides. Mol. CeZZ. BioZ. 10: 6941-6499, 1990. 21. Pacholczyk, T., R. D. Blakely, and S. Amara. Expression cloning of a cocaineand antidepressant-sensitive human noradrenaline transporter. Nature Lond. 350: 350-354, 1991. 22. Pajor, A. M., and E. M. Wright. Cloning and functional expression of a mammalian Na+/nucleoside cotransporter. J. BioZ. Chem. 267: 3557-3560, 1992. 23. Turk, E., B. Zabel, S. Mundlos, J. Dyer, and E. M. Wright. Glucose/galactose malabsorption caused by a defect in the Na+/ glucose cotransporter. Nature Lond. 28: 350, 354-356, 1991. 24. Turner, R. J., and A. Moran. Further studies of proximal tubular brush border membrane D-glucose transport heterogeneity. J. Membr. Biol. 70: 37-45, 1982. 25. Umbach, J. A., M. J. Coady, and E. M. Wright. Intestinal Na+/glucose transporter expressed in Xenopus oocytes is electrogenie. Biophys. J. 57: 1217-1224, 1990. 26. Weber, J. L., and P. E. May. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Human Genet. 44: 388-396, 1989. 27. Werner, A., M. L. Moore, H. Mantei, J. Biber, G. Semenza, and H. Murer. Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc. NcztZ. Acad. Sci. USA 88: 9608-9612, 1991. 11.
Hediger,
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