97
Biochimica et Biophysica Acta, 1132 (1992) 97-99 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90388
Short Sequence-Paper
Nucleotide and primary amino acid sequence of porcine lactoferrin John P. Lydon a, Brid R. O'Malley a Odila Saucedo a, Teresa Lee Denis R. Headon b and Orla M. Conneely a
b
'~ Department of Cell Biology, Baylor College of Medicine, Houston, TX (USA) and b Cell and Molecular Biology Group, Department of Biochemisto,, Unirersity College Galway, Galway (Ireland) (Received 24 April 1992)
Key words: Lactoferrin; Transferrin; Mammary Gland; (Porcine)
A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland Agtll cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.
Lactoferrin was originally detected in human breast milk (1 m g / m l ) [1] and has subsequently been found in other external fluids such as tears, saliva and mucosal secretions [2] and also in neutrophils [3]. As a member of the transferrin family of iron-binding proteins, lactoferrin is a monomeric glycoprotein with bilobal protein structure [4]. Each lobe can bind one ferric atom with high affinity accompanied by the synergistic binding of one bicarbonate molecule [5]. This unique iron uptake system allows lactoferrin to modulate the levels of free iron in a number of biological secretions. Compared to the other transferrins, lactoferrin sequesters iron more tightly and at lower pH values [4]. Based on the iron binding properties of lactoferrin a number of physiological functions have been proposed for this protein. These include antimicrobial functions [7], regulation of intestinal iron absorption in newborns [8], regulation of myelopoiesis [9] and modulation inflammatory responses [10]. A pLF cDNA clone was isolated from an oligo(dT) primed Agtll cDNA library derived from porcine mammary gland tissue using a 1 kb fragment of the human lactoferrin cDNA [11]. The pLF cDNA sequence is shown in Fig. 1. The sequence indicates an open reading frame of 2058 base pairs encoding a protein with a secretion signal peptide of 19 amino acids followed by a mature protein of 667 residues. Furthermore, the pLF amino acid sequence reveals the
Correspondence to: O.M. Conneely, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
bilobal structure exhibited by other members of the transferrin family. The amino acids that appear to coordinate with iron in both lobes of pLF are conserved with respect to previously characterized lactoferrins [4]. A comparison of the amino acid sequence of pLF with the amino acid sequences of bovine [12], human [13] and murine [14] lactoferrins reveals 72.6%, 70.7% and 62.2% amino acid sequence identity, respectively (Fig. 2). References 1 Groves, M.L. (1960) J. Am. Chem. Soc. 82, 3345-3350. 2 Masson, P.L., Heremans. J.F. and Dive, C. (1966) Clin. Chim. Acta. 14, 735-739. 3 Masson, P.L., Heremans, J.F. and Schonne, E. (1969) J. Exp Med. 130, 643-658. 4 Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. and Baker, E.N. (1989) J. Mol. Biol. 209, 711-734. 5 Schlabach, M.R. and Bates, G.W. (1975) J. Biol. Chem. 250, 2182-2188. 6 Nemet, K. and Simonovitis, I. (1985) Haematology 18, 3-12. 7 Arnold, R.R., Cole, M.F. and McGhee, J.R. (1977) 197, 263-265. 8 Hashizume, S., Kuroda, K. and Murakami, M. (1983) Biochim. Biophys. Acta 763, 377-382. 9 Broxemeyer, H.E., DeSousa, M., Smithyman, A., Ralph, P., Hamilton, J., Kurland, J. and Bognacki, N. (1980) Blood 55, 324-333. 10 Oseas, R., Yang, H.H., Baehner, R.L. and Boxer, L.A. (1981) Blood 57, 939-945. 11 Ward, P.P., Lo, J.-Y., Duke, M., May, G.S., Headon, D.R. and Conneely, O.M. (1992) Biotechnology, in press. 12 Pierce, A., Colavizza, D., Benaissa, M., Maes, P., Tartar, A., Montreuil, J. and Spik, G. (1991) Eur. J. Biochem. 196, 177-184. 13 Rey, M.W., Woldshuk, S.L., DeBoer, H.A. and Pieper, F.R. (1990) Nucleic Acid Res. 18, 5288. 14 Pentecost, B.T. and Teng, C.T. (1987) J. Biol. Chem. 262, 87598766.
98
1
TTTCACTGGTTTATGCTTTTATTGCTG~GGAGG~GGGCCCAGAGCAGGAGATGCCAGGCGGGTCCCAGGGGTCCGGGTCCCAG~GGGGTGACCTGGG
101
TCCCAGGCCCTCCTGATTCCGGCTGATGGGGAGGTGGGGTGATATCTTGTCTCCCAGAGGG~CCCCCCGGAGGTGATGGCCGGGACTCCCCTAGAcGCG
201
GCACTGCTCTTCAGCTGGGCTGCAGGATCCGCATGGGCACAGACAGCGTCTTGAGGGCAGTCGCCTCAGGACCTCAGACATG~GCT~TTCATccC~Gcc M K L F I P A
301
CTGCTGTTCCTCTGGACACTTGGACTGTGTCTGGCTGCCCCT~GAAAGGGGTTCGATGGTGTGTCATATCCACAGCAGAGTATT~AAAATGCcGccAGT L L F L W T L G L C L A A P K K G V R W C V I S T A E Y S K C R Q W
401
GGC~TCAAAGAT~G~G~CT~TCCCATCTTCTGCAT~GGAGGGCTTCTCCCACTGACTGTATCCGGGCCATCGCGG~AAAAAGGGCAGATGCTGT Q S K I R R T N P I F C I R R A S P T D C I R A I A A K R A D A V
501
GA~CCTTGATGGTGGTTTGGTGTTTG~GCAGGCCAGTACAAACTGCGGCCGGTAGcAGCGGAGATCTACGGGACAG~GAG~TCCCCAAACCTACTAT T L D G G L V F E A G Q Y K L R P V A A E I Y G T E E N P Q T Y Y
601
TATGCTGTGGCTGTAGTG~GA~GGTTTC~CTTTCAGCTG~CCAGCTAC~GGTCGAAAGTCCTGCCACATAGGcCTTGGCAGGTCTGccGGGTGGA Y A V A V V K K G F N F Q L N Q L Q G R K S C H I G L G R S A G W N
701
ATATCCCTATAGGGTTACTTCGCCGGTTCTTGGACTGGGCAGGGCCACcTGAGCCCCTCCAGAAAGCTGTGGCCAAATTcTTcT•T•AGAG•TGTGTGc• I P I G L L R R F L D W A G P P E P L Q K A V A K F F S Q S C V P
801
cTGCGCAGATGGAAATGCGTATCCC~cCTGTGTCAGCTGTGCATAGGGAAAGGGAAAGATAAATGTGCTTGTTCCTCCCAGG~CCGTATTTTGGcTAT C A D G N A Y P N L C Q L C I G K G K D K C A C S S Q E P Y F G Y
901
TCCGGTGCCTTC~CTGTCTGCACAAAGGGATTGGAGATGTGGCTTTTGTC~GGAGAGTACAGTGTTTGAG~CCTGCCACAG~GGCTGACCGGGAcA S G A F N C L H K G I G D V A F V K E S T V F E N L P Q K A D R D K
1001
~TACGAGCTACTCTGCCCAGAC~TACTCGAAAGCCAGTGG~GCATTCAGG~AGTGCCACCTTGCCCGGGTCCCTTCTCATGCTGTTGTGGCCcG~G Y E L L C P D N T R K P V E A F R E C H L A R V P S H A V V A R S
Ii01
TGTG~TGGC~GGAG~cTCCATCTGGAGCCTTCTCTACCAGTCAcAGAAA~GTTTGGAA~GC~TCCACAGGAGTTCcAGCT~TTTGGcTcT~CT V N G K E N S I W S L L Y Q S Q K K F G K S N P Q E F Q L F G S P
1201
GGTCAGCAG~GGACCTCCTGTTTAGAGACGCTACCATCGGGTTTTTG~GATCCCCTCAAAGATAGATTCT~GCTGTACCTGGGCCTCCCGTA~cTTA G Q Q K D L L F R D A T I G F L K I P S K I D S K L Y L G L P Y L T
1301
cTGCCATCCAGGGCCTGAGGGA~CGGCAGCGGAGGTGGAGGCGCGGCAGGCG~GGTCGTGTGGTGCGCCGTGGGTCCAGAGGAGCTGCGC~GTGCCG A I Q G L R E T A A E V E A R Q A K V V W C A V G P E E L R K C R
1401
GcAGTGGAGCAGCCAGAGCAGCCAG~CCTG~CTGCAGCCTGGCCTCCACCACCGAGGACTGCATCGTCCAGGTGCTGAAAGGAG~GCTGATGCTATG Q W S S Q S S Q N L N C S L A S T T E D C I V Q V L K G E A D A M
1501
AGCTTGGATGGAGGATTTATCTACACTGCGGGC~GTGTGGTTTGGTGCCTGTCCTGGCAGAG~CCAAAAATCTCGCCAAAGCAGTAGCTCAGACTGTG S L D G G F I Y T A G K C G L V P V L A E N Q K S R Q S S S S D C V
!601
TGCATAGACC~CAC~GGGTATTTTGCCGTGGCGGTTGTCAGGAAAGCAAATGGTGGTATCACCTGG~TTCTGTGAGAGGCACG~GTCCTGCCA~AC H R P T Q G Y F A V A V V R K A N G G I T W N S V R G T K S C H T
1701
TGCTGTGGACAGGACAGCAGGCTGG~CATCCCCATGGGCCTGcTTGTC~CCAGACAGGCTCCTGCA~TTTGACG~TTCTTTAGTCAAAGcTGTGcT A V D R T A G W N I P M G L L V N Q T G S C K F D E F F S Q S C A
1801
CcTGGGTCTCAGCCGGGATCC~TCTCTGTGCACTGTGTGTTGGC~TGACCAGGGCGTGGAC~GTGTGTGCCC~CAGT~TGAGAGATACTATGGTT P G S Q P G S N L C A L C V G N D Q G V D K C V P N S N E R Y Y G Y
1901
ACACCGGGGCTTTCAGGTG~CTGGCTGAG~TGCTGGGGATGTGGCGTTTGTGAAAGATGTCACTGTCTTGGAC~CACG~TGGACAG~CACTC~GA T G A F R C L A Z N A G D V A F V K D V T V L D N T N G Q N T Q E
2001
GTGGGCCAGGG~TTGAGGTCAGACGACTTTGAGCTGCTGTGCCTT~TGGCAcCAGG~GCCTGTGACTGAGGCTCAG~CTGTCACCTGGCTGTGGCC W A R E L R S D D F E L L C L N G T R K P V T E A Q N C H L A V A
2101
CCCAGTCATGCTGTGGT~TCTCGG~GGAA~GGCAGCACAGGTAG~CAGATGCTGCTCACTGAGCAGGCTCAGTTTGG~GATAC~GAAAAGACTGcC P S H A V V S R K E K A A Q V Z Q M L L T E Q A Q F G R Y G K D C P
2201
cAGAC~GTTTTGCTTGTT~CGGTCTGAGACCAAAAACCTTCTGTTC~CGAC~CACGGAGTGTCTGGCCC~CTC~GGCAA~C~CATACGAAAA D K F C L F R S E T K N L L F N D N T E C L A Q L Q G K T T Y E K
2301
ATATTTGGGATCAGAGTATGTCACAGCCATCGCTACCTGAAACATGCTGCAGCCTCCCCTCAGTCTGG~GCCTGTGCCTTCATGATGAGGTAAAAcCGG Y L G S E Y V T A I A T *
2401
AAAAG~GCTGCCCCGCCTCCCCAGGGGCCTCAGCTTTCCCTCCTC~CGTCTTGATTCCCAGCTGCCCTGGGCCTGCCTCTCTCCCTTCCTGAGGGCAGA
2501
CTTTGTTCAGCTCATCCGTTTTCAC~TTCCCTGCTGGCCTCTTAGC~GAAATAA~TTAGAAATTCTGCCG~TTC
Fig. 1. Nucleotide and predicted amino acid sequence of PLF.
99 UPPER
CASE
-
aligned
lower
case
=
unaligned
........... ..........
spplfg bovlactra humltfrg musult
spplfg bovlactra humltfrg musult
spplfg bovlactra humltfrg musult
spplfg
non-identical
aligned -
identical
gap
:
MKLFIPALLFLWTLGLCLA.APKKGVRWCVISTAEYSKCRQWQSKIR..R..TNPIFCIR
:
....
:
---VFLV
:
IQGLLIPS-IFLEALGLCL.-KATT
V ....
S-GA
....
......
.--R-N
GA ......
....
T--QP-WF---R--WRMK..KIgAPS-T-V-
gRRRRS-Q--AV-QP-AT--F---RNM-kv-..GP-VS--K ....
AV-NS-EE--LR--NEM-..KvgGP-LS-VK
:
RASPTDCIRAIAAKRADAVTLDGGLVFEA..GQYKLRPVAAEIYGTEENPQTYYYAVAVV
:
--FALE
:
-D--IQ--Q---EN
..........
FIY--glAP
.........
V ....
:
KS-TRQ--Q--VTN
....
TM-D-gkPP
.........
V---K-Q-R-H
......
E-K
.........
M ....
M .....
grDP
.............
:
KKGFNFQLNQLQGRKSCHIGLGRSAGWNIPIGLLRRFLDWAGPPEPLQKAVAKFFSQSCV
:
---S
:
---GS
:
-NSS--H
....
D .........
....
E---L
......
T ........
....
T--R-T
LR---T-I
......
V-T-T--P--N-T K ....
PCADGNAYPNLCQLCIGKGKDKCACSSQEPYFGYSGAFNCLHKGIGDVAFVKESTVFENL --I-RQ
humltfrg
:
-G--KGQF
....
R--A-T-EN---F
musult
:
-G-QKDRF
....
SS-A-T-AN---S-PE---S--A--LR--RDNA
spplfg
:
PQKADRDKYELLCPDNTRKPVEAFRECHLARVPSHAVVARSVNGKENSIWSLLYQSQKKF
:
-E .....
:
SDE-E--E
musult
:
-N--E--Q-K
spplfg
bovlactra humltfrg
Q .....
LN-S-A--D--K
.......
R ..........
.......
.............
..... DK-KD
S .....
Q .......
........ .....
IR ......
D-
.....
TRG
E-
DA--N--R-A-E--
GKSNPQEFQLFGSPGQQKDLLFRDATIGFLKIPSKIDSKLYLGLPYLTAIQGLRETAAEV --NKSRS
.......
PG-R
humltfrg
:
--DKSPK
.......
SG ......
musult
:
--KQASG
....
spplfg
:
EARQAKVVWCAVGPEELRKCRQWSSQSSQNLNCSLASTTEDCIVQVLKGEADAMSLDGGF
:
K--YTR
..........
:
A--R-R
.......
musult
:
I-SK-R-T
spplfg
:
IYTAGKCGLVPVLAENQKSRQSS..SSDCVHRPTQGYFAVAVVRKANGGITWNSVRGTKS
bovlactra humltfrg
bovlactra
A--SG
.....
V--A---ASR---TLKN
K-SA---SRV-PR---G
......
....
....
N---GL-EGSVT--S
D ....
........
S--K---D--NRD-RGRVT-ISFP
................
,--,,,-..-,---,---,--,
.....
:
V---C
,--,---,,,,,--,--,--,
......
musult
:
...................
spplfg
:
CHTAVDRTAGWNIPMGLLVNQTGSCKFDEFFSQSCAPGSQPGSNLCALCVGNDQGVDKCV
:
.................
:
..................
F ..........
musult
:
..................
A---R
spplfg
:
PNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLDNTNGQNTQEWARELRSDDFELLCLNG
bovlactra humltfrg
...........
I .......
....
---K-K
musult
:
---K---Q
spplfg
:
TRKPVTEAQNCHLAVAPSHAVVSRKEKAAQVEQMLLTEQAQFGRYGKDCPDKFCLFRSET
spplfg
humltfrg musult
L .....
S .......
ND--WE
AD-K
.................................
bovlactra
DV .......
N ..........
D-R
:
musult
L ........
AIM--D
LN ....
Y Y
.........
K---E-L
....
RSDTSL
RD-K-R
Y .........
humltfrg
humltfrg
.............
A ............
:
bovlactra
E--
N--KSEE--
Y
LKDK--
.....
K-K--
,,-,..,,---,--,--,---,--,,,-,--,-,--,--
bovlactra
.....
....
AL ..............
.......
:
humltfrg
.....
SG-F
KESA---VRV-Q-V-VG---TFS-T-S--N-NKKQQD-
QK--Q---Q--G--VT-AT EQ .....
.....
,--,-,--,--,--,---,--
:
....
T ......
D---DL--K--SKA-E--
:
K-SAL---R
K---
K--RD-A
bovlactra
....
....
K--QD-A
Q ...........
.....
A---
IEA---R---A---
....................
W---TEYK
.......
T--PY-N-N---AS-EE--S
:
.....
.......
.......
.....
:
K-E-ENQ
.......
RQ-R-H
V--M-I--PY-S-TESL--PPG
....
bovlactra
.........
K-S---H
....
A-D---L
.......
I-DEK-EN--A
ES-AD--KN-NRE--R
....
:
.........
:
K .......
RS ....
M--N
......
MD-VERLK-V--HQ--K---N-S
:
........
K .....
I--N
......
TD-VEVLQ-VV-DQ-V
N-KLK
SDR--H-K-V--HQ--L--KN--N
....
D-
....
D-
.......
........ ......... ....
:
KNLLFNDNTECLAQLQGKTTYEKYLGSEYVTAIAT
:
.............
K-G-RP---E---T
:
.............
R~H
..........
PQ--AG-TNLKKCSTSPLLEACEFLRK
:
.............
KIP
....
K---I-TERLKQCSSSPLLEACAFLTQ
S .....
....
I-DE--EN---
Q--D-N-NEA--KD-KLA--A
,--,---,--,---,--,-,--,
N ......
..... .......
N-QR--GE
....
DD
K--Q--Q-K-
.................. .......
NLKKCSTSPLLEACAFLTR
Fig. 2. Alignmentofthe amino acid sequence ofporcinelactoferrin(spplfg) withthe bovine(bovlactra),human(humltfrg)and murine(musult) lacto~rrin.
Biochimica et Biophysica Acta, 1132 (1992) 97-99 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90388
Short Sequence-Paper
Nucleotide and primary amino acid sequence of porcine lactoferrin John P. Lydon a, Brid R. O'Malley a Odila Saucedo a, Teresa Lee Denis R. Headon b and Orla M. Conneely a
b
'~ Department of Cell Biology, Baylor College of Medicine, Houston, TX (USA) and b Cell and Molecular Biology Group, Department of Biochemisto,, Unirersity College Galway, Galway (Ireland) (Received 24 April 1992)
Key words: Lactoferrin; Transferrin; Mammary Gland; (Porcine)
A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland Agtll cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.
Lactoferrin was originally detected in human breast milk (1 m g / m l ) [1] and has subsequently been found in other external fluids such as tears, saliva and mucosal secretions [2] and also in neutrophils [3]. As a member of the transferrin family of iron-binding proteins, lactoferrin is a monomeric glycoprotein with bilobal protein structure [4]. Each lobe can bind one ferric atom with high affinity accompanied by the synergistic binding of one bicarbonate molecule [5]. This unique iron uptake system allows lactoferrin to modulate the levels of free iron in a number of biological secretions. Compared to the other transferrins, lactoferrin sequesters iron more tightly and at lower pH values [4]. Based on the iron binding properties of lactoferrin a number of physiological functions have been proposed for this protein. These include antimicrobial functions [7], regulation of intestinal iron absorption in newborns [8], regulation of myelopoiesis [9] and modulation inflammatory responses [10]. A pLF cDNA clone was isolated from an oligo(dT) primed Agtll cDNA library derived from porcine mammary gland tissue using a 1 kb fragment of the human lactoferrin cDNA [11]. The pLF cDNA sequence is shown in Fig. 1. The sequence indicates an open reading frame of 2058 base pairs encoding a protein with a secretion signal peptide of 19 amino acids followed by a mature protein of 667 residues. Furthermore, the pLF amino acid sequence reveals the
Correspondence to: O.M. Conneely, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
bilobal structure exhibited by other members of the transferrin family. The amino acids that appear to coordinate with iron in both lobes of pLF are conserved with respect to previously characterized lactoferrins [4]. A comparison of the amino acid sequence of pLF with the amino acid sequences of bovine [12], human [13] and murine [14] lactoferrins reveals 72.6%, 70.7% and 62.2% amino acid sequence identity, respectively (Fig. 2). References 1 Groves, M.L. (1960) J. Am. Chem. Soc. 82, 3345-3350. 2 Masson, P.L., Heremans. J.F. and Dive, C. (1966) Clin. Chim. Acta. 14, 735-739. 3 Masson, P.L., Heremans, J.F. and Schonne, E. (1969) J. Exp Med. 130, 643-658. 4 Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. and Baker, E.N. (1989) J. Mol. Biol. 209, 711-734. 5 Schlabach, M.R. and Bates, G.W. (1975) J. Biol. Chem. 250, 2182-2188. 6 Nemet, K. and Simonovitis, I. (1985) Haematology 18, 3-12. 7 Arnold, R.R., Cole, M.F. and McGhee, J.R. (1977) 197, 263-265. 8 Hashizume, S., Kuroda, K. and Murakami, M. (1983) Biochim. Biophys. Acta 763, 377-382. 9 Broxemeyer, H.E., DeSousa, M., Smithyman, A., Ralph, P., Hamilton, J., Kurland, J. and Bognacki, N. (1980) Blood 55, 324-333. 10 Oseas, R., Yang, H.H., Baehner, R.L. and Boxer, L.A. (1981) Blood 57, 939-945. 11 Ward, P.P., Lo, J.-Y., Duke, M., May, G.S., Headon, D.R. and Conneely, O.M. (1992) Biotechnology, in press. 12 Pierce, A., Colavizza, D., Benaissa, M., Maes, P., Tartar, A., Montreuil, J. and Spik, G. (1991) Eur. J. Biochem. 196, 177-184. 13 Rey, M.W., Woldshuk, S.L., DeBoer, H.A. and Pieper, F.R. (1990) Nucleic Acid Res. 18, 5288. 14 Pentecost, B.T. and Teng, C.T. (1987) J. Biol. Chem. 262, 87598766.
98
1
TTTCACTGGTTTATGCTTTTATTGCTG~GGAGG~GGGCCCAGAGCAGGAGATGCCAGGCGGGTCCCAGGGGTCCGGGTCCCAG~GGGGTGACCTGGG
101
TCCCAGGCCCTCCTGATTCCGGCTGATGGGGAGGTGGGGTGATATCTTGTCTCCCAGAGGG~CCCCCCGGAGGTGATGGCCGGGACTCCCCTAGAcGCG
201
GCACTGCTCTTCAGCTGGGCTGCAGGATCCGCATGGGCACAGACAGCGTCTTGAGGGCAGTCGCCTCAGGACCTCAGACATG~GCT~TTCATccC~Gcc M K L F I P A
301
CTGCTGTTCCTCTGGACACTTGGACTGTGTCTGGCTGCCCCT~GAAAGGGGTTCGATGGTGTGTCATATCCACAGCAGAGTATT~AAAATGCcGccAGT L L F L W T L G L C L A A P K K G V R W C V I S T A E Y S K C R Q W
401
GGC~TCAAAGAT~G~G~CT~TCCCATCTTCTGCAT~GGAGGGCTTCTCCCACTGACTGTATCCGGGCCATCGCGG~AAAAAGGGCAGATGCTGT Q S K I R R T N P I F C I R R A S P T D C I R A I A A K R A D A V
501
GA~CCTTGATGGTGGTTTGGTGTTTG~GCAGGCCAGTACAAACTGCGGCCGGTAGcAGCGGAGATCTACGGGACAG~GAG~TCCCCAAACCTACTAT T L D G G L V F E A G Q Y K L R P V A A E I Y G T E E N P Q T Y Y
601
TATGCTGTGGCTGTAGTG~GA~GGTTTC~CTTTCAGCTG~CCAGCTAC~GGTCGAAAGTCCTGCCACATAGGcCTTGGCAGGTCTGccGGGTGGA Y A V A V V K K G F N F Q L N Q L Q G R K S C H I G L G R S A G W N
701
ATATCCCTATAGGGTTACTTCGCCGGTTCTTGGACTGGGCAGGGCCACcTGAGCCCCTCCAGAAAGCTGTGGCCAAATTcTTcT•T•AGAG•TGTGTGc• I P I G L L R R F L D W A G P P E P L Q K A V A K F F S Q S C V P
801
cTGCGCAGATGGAAATGCGTATCCC~cCTGTGTCAGCTGTGCATAGGGAAAGGGAAAGATAAATGTGCTTGTTCCTCCCAGG~CCGTATTTTGGcTAT C A D G N A Y P N L C Q L C I G K G K D K C A C S S Q E P Y F G Y
901
TCCGGTGCCTTC~CTGTCTGCACAAAGGGATTGGAGATGTGGCTTTTGTC~GGAGAGTACAGTGTTTGAG~CCTGCCACAG~GGCTGACCGGGAcA S G A F N C L H K G I G D V A F V K E S T V F E N L P Q K A D R D K
1001
~TACGAGCTACTCTGCCCAGAC~TACTCGAAAGCCAGTGG~GCATTCAGG~AGTGCCACCTTGCCCGGGTCCCTTCTCATGCTGTTGTGGCCcG~G Y E L L C P D N T R K P V E A F R E C H L A R V P S H A V V A R S
Ii01
TGTG~TGGC~GGAG~cTCCATCTGGAGCCTTCTCTACCAGTCAcAGAAA~GTTTGGAA~GC~TCCACAGGAGTTCcAGCT~TTTGGcTcT~CT V N G K E N S I W S L L Y Q S Q K K F G K S N P Q E F Q L F G S P
1201
GGTCAGCAG~GGACCTCCTGTTTAGAGACGCTACCATCGGGTTTTTG~GATCCCCTCAAAGATAGATTCT~GCTGTACCTGGGCCTCCCGTA~cTTA G Q Q K D L L F R D A T I G F L K I P S K I D S K L Y L G L P Y L T
1301
cTGCCATCCAGGGCCTGAGGGA~CGGCAGCGGAGGTGGAGGCGCGGCAGGCG~GGTCGTGTGGTGCGCCGTGGGTCCAGAGGAGCTGCGC~GTGCCG A I Q G L R E T A A E V E A R Q A K V V W C A V G P E E L R K C R
1401
GcAGTGGAGCAGCCAGAGCAGCCAG~CCTG~CTGCAGCCTGGCCTCCACCACCGAGGACTGCATCGTCCAGGTGCTGAAAGGAG~GCTGATGCTATG Q W S S Q S S Q N L N C S L A S T T E D C I V Q V L K G E A D A M
1501
AGCTTGGATGGAGGATTTATCTACACTGCGGGC~GTGTGGTTTGGTGCCTGTCCTGGCAGAG~CCAAAAATCTCGCCAAAGCAGTAGCTCAGACTGTG S L D G G F I Y T A G K C G L V P V L A E N Q K S R Q S S S S D C V
!601
TGCATAGACC~CAC~GGGTATTTTGCCGTGGCGGTTGTCAGGAAAGCAAATGGTGGTATCACCTGG~TTCTGTGAGAGGCACG~GTCCTGCCA~AC H R P T Q G Y F A V A V V R K A N G G I T W N S V R G T K S C H T
1701
TGCTGTGGACAGGACAGCAGGCTGG~CATCCCCATGGGCCTGcTTGTC~CCAGACAGGCTCCTGCA~TTTGACG~TTCTTTAGTCAAAGcTGTGcT A V D R T A G W N I P M G L L V N Q T G S C K F D E F F S Q S C A
1801
CcTGGGTCTCAGCCGGGATCC~TCTCTGTGCACTGTGTGTTGGC~TGACCAGGGCGTGGAC~GTGTGTGCCC~CAGT~TGAGAGATACTATGGTT P G S Q P G S N L C A L C V G N D Q G V D K C V P N S N E R Y Y G Y
1901
ACACCGGGGCTTTCAGGTG~CTGGCTGAG~TGCTGGGGATGTGGCGTTTGTGAAAGATGTCACTGTCTTGGAC~CACG~TGGACAG~CACTC~GA T G A F R C L A Z N A G D V A F V K D V T V L D N T N G Q N T Q E
2001
GTGGGCCAGGG~TTGAGGTCAGACGACTTTGAGCTGCTGTGCCTT~TGGCAcCAGG~GCCTGTGACTGAGGCTCAG~CTGTCACCTGGCTGTGGCC W A R E L R S D D F E L L C L N G T R K P V T E A Q N C H L A V A
2101
CCCAGTCATGCTGTGGT~TCTCGG~GGAA~GGCAGCACAGGTAG~CAGATGCTGCTCACTGAGCAGGCTCAGTTTGG~GATAC~GAAAAGACTGcC P S H A V V S R K E K A A Q V Z Q M L L T E Q A Q F G R Y G K D C P
2201
cAGAC~GTTTTGCTTGTT~CGGTCTGAGACCAAAAACCTTCTGTTC~CGAC~CACGGAGTGTCTGGCCC~CTC~GGCAA~C~CATACGAAAA D K F C L F R S E T K N L L F N D N T E C L A Q L Q G K T T Y E K
2301
ATATTTGGGATCAGAGTATGTCACAGCCATCGCTACCTGAAACATGCTGCAGCCTCCCCTCAGTCTGG~GCCTGTGCCTTCATGATGAGGTAAAAcCGG Y L G S E Y V T A I A T *
2401
AAAAG~GCTGCCCCGCCTCCCCAGGGGCCTCAGCTTTCCCTCCTC~CGTCTTGATTCCCAGCTGCCCTGGGCCTGCCTCTCTCCCTTCCTGAGGGCAGA
2501
CTTTGTTCAGCTCATCCGTTTTCAC~TTCCCTGCTGGCCTCTTAGC~GAAATAA~TTAGAAATTCTGCCG~TTC
Fig. 1. Nucleotide and predicted amino acid sequence of PLF.
99 UPPER
CASE
-
aligned
lower
case
=
unaligned
........... ..........
spplfg bovlactra humltfrg musult
spplfg bovlactra humltfrg musult
spplfg bovlactra humltfrg musult
spplfg
non-identical
aligned -
identical
gap
:
MKLFIPALLFLWTLGLCLA.APKKGVRWCVISTAEYSKCRQWQSKIR..R..TNPIFCIR
:
....
:
---VFLV
:
IQGLLIPS-IFLEALGLCL.-KATT
V ....
S-GA
....
......
.--R-N
GA ......
....
T--QP-WF---R--WRMK..KIgAPS-T-V-
gRRRRS-Q--AV-QP-AT--F---RNM-kv-..GP-VS--K ....
AV-NS-EE--LR--NEM-..KvgGP-LS-VK
:
RASPTDCIRAIAAKRADAVTLDGGLVFEA..GQYKLRPVAAEIYGTEENPQTYYYAVAVV
:
--FALE
:
-D--IQ--Q---EN
..........
FIY--glAP
.........
V ....
:
KS-TRQ--Q--VTN
....
TM-D-gkPP
.........
V---K-Q-R-H
......
E-K
.........
M ....
M .....
grDP
.............
:
KKGFNFQLNQLQGRKSCHIGLGRSAGWNIPIGLLRRFLDWAGPPEPLQKAVAKFFSQSCV
:
---S
:
---GS
:
-NSS--H
....
D .........
....
E---L
......
T ........
....
T--R-T
LR---T-I
......
V-T-T--P--N-T K ....
PCADGNAYPNLCQLCIGKGKDKCACSSQEPYFGYSGAFNCLHKGIGDVAFVKESTVFENL --I-RQ
humltfrg
:
-G--KGQF
....
R--A-T-EN---F
musult
:
-G-QKDRF
....
SS-A-T-AN---S-PE---S--A--LR--RDNA
spplfg
:
PQKADRDKYELLCPDNTRKPVEAFRECHLARVPSHAVVARSVNGKENSIWSLLYQSQKKF
:
-E .....
:
SDE-E--E
musult
:
-N--E--Q-K
spplfg
bovlactra humltfrg
Q .....
LN-S-A--D--K
.......
R ..........
.......
.............
..... DK-KD
S .....
Q .......
........ .....
IR ......
D-
.....
TRG
E-
DA--N--R-A-E--
GKSNPQEFQLFGSPGQQKDLLFRDATIGFLKIPSKIDSKLYLGLPYLTAIQGLRETAAEV --NKSRS
.......
PG-R
humltfrg
:
--DKSPK
.......
SG ......
musult
:
--KQASG
....
spplfg
:
EARQAKVVWCAVGPEELRKCRQWSSQSSQNLNCSLASTTEDCIVQVLKGEADAMSLDGGF
:
K--YTR
..........
:
A--R-R
.......
musult
:
I-SK-R-T
spplfg
:
IYTAGKCGLVPVLAENQKSRQSS..SSDCVHRPTQGYFAVAVVRKANGGITWNSVRGTKS
bovlactra humltfrg
bovlactra
A--SG
.....
V--A---ASR---TLKN
K-SA---SRV-PR---G
......
....
....
N---GL-EGSVT--S
D ....
........
S--K---D--NRD-RGRVT-ISFP
................
,--,,,-..-,---,---,--,
.....
:
V---C
,--,---,,,,,--,--,--,
......
musult
:
...................
spplfg
:
CHTAVDRTAGWNIPMGLLVNQTGSCKFDEFFSQSCAPGSQPGSNLCALCVGNDQGVDKCV
:
.................
:
..................
F ..........
musult
:
..................
A---R
spplfg
:
PNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLDNTNGQNTQEWARELRSDDFELLCLNG
bovlactra humltfrg
...........
I .......
....
---K-K
musult
:
---K---Q
spplfg
:
TRKPVTEAQNCHLAVAPSHAVVSRKEKAAQVEQMLLTEQAQFGRYGKDCPDKFCLFRSET
spplfg
humltfrg musult
L .....
S .......
ND--WE
AD-K
.................................
bovlactra
DV .......
N ..........
D-R
:
musult
L ........
AIM--D
LN ....
Y Y
.........
K---E-L
....
RSDTSL
RD-K-R
Y .........
humltfrg
humltfrg
.............
A ............
:
bovlactra
E--
N--KSEE--
Y
LKDK--
.....
K-K--
,,-,..,,---,--,--,---,--,,,-,--,-,--,--
bovlactra
.....
....
AL ..............
.......
:
humltfrg
.....
SG-F
KESA---VRV-Q-V-VG---TFS-T-S--N-NKKQQD-
QK--Q---Q--G--VT-AT EQ .....
.....
,--,-,--,--,--,---,--
:
....
T ......
D---DL--K--SKA-E--
:
K-SAL---R
K---
K--RD-A
bovlactra
....
....
K--QD-A
Q ...........
.....
A---
IEA---R---A---
....................
W---TEYK
.......
T--PY-N-N---AS-EE--S
:
.....
.......
.......
.....
:
K-E-ENQ
.......
RQ-R-H
V--M-I--PY-S-TESL--PPG
....
bovlactra
.........
K-S---H
....
A-D---L
.......
I-DEK-EN--A
ES-AD--KN-NRE--R
....
:
.........
:
K .......
RS ....
M--N
......
MD-VERLK-V--HQ--K---N-S
:
........
K .....
I--N
......
TD-VEVLQ-VV-DQ-V
N-KLK
SDR--H-K-V--HQ--L--KN--N
....
D-
....
D-
.......
........ ......... ....
:
KNLLFNDNTECLAQLQGKTTYEKYLGSEYVTAIAT
:
.............
K-G-RP---E---T
:
.............
R~H
..........
PQ--AG-TNLKKCSTSPLLEACEFLRK
:
.............
KIP
....
K---I-TERLKQCSSSPLLEACAFLTQ
S .....
....
I-DE--EN---
Q--D-N-NEA--KD-KLA--A
,--,---,--,---,--,-,--,
N ......
..... .......
N-QR--GE
....
DD
K--Q--Q-K-
.................. .......
NLKKCSTSPLLEACAFLTR
Fig. 2. Alignmentofthe amino acid sequence ofporcinelactoferrin(spplfg) withthe bovine(bovlactra),human(humltfrg)and murine(musult) lacto~rrin.
