Biochimica et BiophysicaActa, ! 121 (1992)83-87 © 1992 Elsevier Science Publishers B.V. All rights resep.,ed 0167-4838/92/$05.00
83
BBAPRO 34194
Molecular cloning and characterization of mouse mast cell chymases Wei Chu 1, David A. Johnson and Phillip R. Musich Department of Biochemistry, James H. QuiUenCollegeof Medicine, East TennesseeState Unicersity,Johnson City, TN (USA)
(Received 7 August 1991) (Revised manuscript received 11 December 1991)
Key words: cDNA; Mast cell; Proteinase; Amino acid seq,-.nce
Mouse mast cell chymases are granule-associated serine proteinases with chymotrypsin-like substrate specificities, cDNAs for two new chymases were isolated from a cDNA library constructed using mRNA from ABFTL-6 mouse mast cells by screening with a rat mast cell proteinase cDNA. The deduced amino acid sequence of mouse chymase 1 consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. Chymase I is unusual in that an Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine proteinase. Also, chymase 1 is expected to have a large positive charge ( + 13) at physiological pH. A partial cDNA for chymase 2 encodes 177 residues of the carboxy terminal portion of a second proteinase distinct from chymase I. Chymase 2 cDNA contains a highly conserved intron/exon junction, a high positive charge ( + 17) and a novel, second potential N-glycosylation site. Tt,mscripts for both chymases are found in ABFTL-6 mast cells, but only chymase 2 mRNA is in mouse connectk,e tissue mast cells. These data suggest that these chymases have distinct enzymatic properties and tissue-specific patterns of gene expression.
Introduction Mast cells contain granule-associated proteolytic enzymes with both trypsin-like and chymotrypsin-like specificities, referred to as tryptase and chymase, respectively. In the mouse, six distinct chymotryptic proteinases (denoted as MMCP-I through MMCP-6) have been identified in mast cells based on partial sequencing of their amino termini [1]. Complete sequences for several of these chymases have been determined either from the purified proteins or based on the nucleotide sequences of their cloned cDNAs. The first chymase, mouse mast cell proteinase (MMCP)-I, was isolated from mice infected with Trichinefla spiralis [2] and its amino acid sequence was determined [3]. Subsequently, a MMCP-2 cDNA was identified from Kirsten sarcoma virus-immortalized mast cells [4]. Recently, sequences
t Present address: Molecular Genetics Department, Hoffman-La Roche, Nutley, N J, USA. Abbreviations: cDNA, complementary DNA; kb, kilobascs; MMCP, mouse mast cell proteinase; ORF, open reading frame; RMCP, rat mast cell pmteinase. This article has been submitted to GENBANK; accession numbers: chymase 1, m68898; chymase 2, M68899. Correspondence: P.R. Musich, Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0581, USA.
for chymase MMCP-4 have been reported [5]. ABFTL-6 mouse mast cells, an Abelson virus-transformed line [6], contain R N A homologous to rat mast cell proteinase (RMCP) II cDNA [7]. Thus, the chymotryptic composition of mouse mast cells may depend on the specific mast cell type, its tissue micro-environment and its state of activation. This report describes the cloning, characterization and transcriptional analysis of complementary DNAs (cDNAs) encoding mouse mast cell chymases different from those previously reported. The deduced amino acid sequences of these chymases indicate novel substrate preferences, ionic interactions and secondary modifications which distinguish them from other, known chyrnases.
Materials and Methods R N A isolation and cDNA library construction. ABFFL-6 mast cells were grown as described [6] and their R N A isolated by the guanidine thiocyanate/CsCI density gradient centrifugation method [8]. Poly(A) + RNA was purified by two cycles of chromatography on oligo(dT)-cellulose [9]. The c D N A library was prepared using a Pharmacia cDNA synthesis kit and Stratagene Agtl0 cloning and Gigapack packaging kits. Positive clones were identified by hybridization screening of
84 plaque lifts of the unamplified library with RMCP II eDNA probe to isolate clones AC1 through AC9. The library was rescreened with the insert of the recombinant ,~C3 identified in the first round to isolate additional positive clones. Hybridizations were performed under standard conditions [10] at 42°C in a buffer containing 0.45 M NaCI, 0.045 M sodium citrate, 10 mM Tris-HCI (pH 7.4), 0.2% sodium dodecyl sulfate, 2% non-fat dry milk and 50% formamide, followed by washes at 65°C in 0.45 M NaCI-0.045 M sodium citrate containing 0.1% sodium dodecyl sulfate. These conditions require the stable hybrids to have 86% sequence identity to mouse chymase eDNA (49% G + C [11]). DNA probes were labeled with [32p]dATP by the random primer method [12]. Isolation and sequencing of recombinants. DNA from plaque-purified phage was isolated [13] and the cDNA inserts and their fragments subcloned into pTZI9U plasmid for sequence analysis by the dideoxy chain termination method using the Sequenase TM protocol (United States Biochemicals). Data were analyzed using Hitachi DNASIS/PROSIS software.
Mouse Chymue 2
Mouse ChymMe 1
L ~ pxC26
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p~C.St. 1 t_1.
(A)n
4: 1--
?--
i
L i
• I
p),cst
i
(A)n I
re
p~-Ct9
I1
14 chymase clones were selected for sequence analysis. The nucleotide sequence for chymase 1, compiled from clones p,~C19, 26 and 51.1, and its derived amino acid sequence are shown in Fig. 1A and B. The cDNA is 960 bp long and contains a 744 bp open reading frame (ORF) adjacent to 5' and 3' untranslated regions of 5 and 211 bases, respectively. The translated sequence consists of 247 amino acids including a hydrophobic signal peptide of 21 amino acids. Mature mouse chymase 1 contains 226 amino acids with a calculated M r of 25.4 kDa, equal to that of dog mast cell chymase [14], MMCP-I [3] and MMCP-4 [5], but two amino acids longer than MMCP-2 [4]. Mouse chymase 1 exhibits many features characteristic of serine proteinases (Fig. 2), including the highly conserved residues comprising the catalytic 'charge-relay' system corresponding to the His-57, Asp-102 and Set-195 in chymotrypsin. Its sequence identity ranges from 72% with dog chymase to 34% with bovine chymotrypsin. The amino-terminal sequence data of
C.
1 l
p~.C102 mm
Results and Discussion
_ } - -
(p×c ~- 9)
B.
I
-20 -I0 H H L L T L H L L L L L L G S S T K ~GR~ATC~T C ~ CT TAC ~i~ ~ C A T ~ C e T T C T ~ CT~TTt~-AG CACC~
-i *I A G E I T ~ AG AG AT
?1
10 20 I G G T E C l P H S R P Y M A ¥ L E I V T S E CATTGGAGGCACGGAG TGCATACCACAC'1'CCCGCCCCTACATGGCCTATCTGGAAATTGTC &CTT ~ G A G
141
30 40 N Y U S A C S G • L ~ R R N F V L T & A H ~---JL~C C~GT CGG¢C'IGP . J ~ . G C ] ~ CCTGATRJ~, J ~ G ~ G W I " G CIGA CTGC~ ~ ~
211
50 * 60 70 R S Z T V L L G & H N K T S K S D T W Q K r_ E G~ G G T C C A T ~ C A 6 T C C I C C I ~ k G G A G C C C A T ~ C ~ C J 4 T CTRA~GJUtGACACGT~CAG ~ G ~ G A
281
80 • 90 V E R Q F L H P K • D E N L V V H D i M L GGTGGRAkI~c ~A'rCCCI'TCATCCItA,kATATG&TG WJ &I~TTTGGTTGTCCACGAC~T CATG~
351
1O0 110 L X E K A K L T L G V G T L P L S A N F N l:' I P ~ G A~G G&G.MIUIK;CCiM~GC~AACCC~AGGTG~X~GG~ACCCCCCC~CTCTCTGCC~ c ~ r CAN: ~ TR TC C
421
120 130 140 P G R Iq C R & V O W G R T N V N E P & S D T L C&CCCGGGAG~J4TGTGC ~G(;CAGTTGGC1T~.GG CAGAACk~.CG ¢GPJ~TGAGCCAGC~ ~ G A C A C A ~
491
150 160 Q it Y R M R L Q E P '~ A C K H F T S F R H N S ~GT;tAN;&~;N;&CTCCAGGRG L'CCCRd~ CCI~GC~CRCI~CACCRGT ~ T C ~ C A C ~ C C
561
170 i 180 • 9 L c V G el IP K K M Q N v Y K G D s G G P ~ CAOCT'GTGTGTGGGCAACCCC,g,&OF.AOA'J~CAAAAI'GTATACAA(~GAG&CTCTGGAGGRC~~
fi31
190 200 210 A G X A Q G X A S Y q H R N M K P P A V F T R GTG~I"RGC~R~,C&T~GCAT¢CZWrGTACATCGGR.R'~CG&RAGCCCCCTGCTGT~ ~ R C ~ G
701 771 841 911
22O I S H • R P W I N It I L R E H End RRTCTCC~TTAC~C CeT~TCR~TRAGATCTTGA~OAATTARCT~G GAG ~ G C ~ GTGAGGAAATCTGGRACTGGRJ4T>GCAGGTTTTGTGTG CC&TGCG&TCTGGC ~ T ~ G T ~ C ~ C1~GARG4:CCIX;CC~GGTCC CeGRGC~CCAGRAGG~r C T e A C P I ~ G T C A C A G ~ A ~ C ~ C ~ C C ATCTTC~TT J ~ C C T ~ G & ~ T T GTCA.q.CTGC~ 960
C
A
G
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L G
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71
50 ~q M T V T T GAG'I'RACA1~ ATG"I"CRTGORC,C1~rc-GRRGAGRAGI~G~ ' R ' I ~ " [ ~ 4 ~ I ~ ~ R C T G ~
141
60 70 80 H N V R K R E C T Q ~ K I K V E K 1" I L, P P N TCAT~TGTGAGAAAGAGAGARTGCACA~ CAGAJ~GATRAR~GTI"GRJM~GTACATCTTGCCTCC~T
211
. • 90 100 Y N V S S K F N D I V L L K L E K Q & M,~ L T $ A TAC.ARTGTGTC~C~GTTCARI~I;RCATCGTA~Z'ACTGRA~"]'GAP.ARGCARGCTJ~"n'GA~
28]
]10 120 V D V V P L P A P S D F A K E> G T M C W A & G CTGTGGATG TAGT TCCCCTGCC~GCCCC CTCTG&CT~r~G;CCRJ4GCC~GGGACGATGCGC ' J ~ , G G C A G ~
35]
130 W G R T G L K X S CTG GGC,G CG R A C T G G A T T G A ~ T
421
K K ~ G G
491
A V Y ~ G D G G P L L C A G V R N G I V $ TGG.CRTCAGTAT~C ATGGGAGRCTCTGGAGGACCTCTR.CTGTGTGC T C . G G G T G G C C C , ~ . ~ T A ~ T A ~
561
200 210 220 S G R G N A K P P A I F T R I S P H V P kl I N TT CTGGACGCGGRJ~ATGCF_%~GCCCCCTGCRJ~'~J~CRC C C C ~ T C T C C C C A C & T G ~ ; C C C I G G & ~ T ~
631 701 771 841
226 R V l E G K End AGAGTCA'~AGAGGGCAAGTAGTGAAR~GCCTGACCTGCGTGCATCAGAGTCTTCAAGCCAGAGCTCTT~ G & T A A C C C T T G G G T TC RR C A J ~ A T G T G T CCAT C C ~ T CCCCTGCCTGC(~CC~A~?J%TG T C C t ' T ~ A T G T C C C C C G C C TG CC C T C A C ~ G C C C C C A G T C TGTCCCCRJ%GATG&~TAJ4AGTCTGT~ &TGA T G G A C C G T T C C C T G T ~ 901
A
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140 I S R T L R E q E L A T C T C & C G T R C C C T G A G ~G R~G T A G A A ~
T
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150 R ~1 N G R G ~ & T ~
150 170 C K I F K H Y K D $ I. ~ I C V G S S C C T G T A ~ J % A T A T T T A R G C & T T R C R A G G A T A G C C T C C ~ G AT C T G ~ G ~ G T T
T K C ~
V
Fig. 1. (A) Subcloning and sequencing strategy for mouse chymase 1 and 2 cDNAs. The rectangles repre~nts ORFs. The speckled, open and shaded areas repre~nt the putative signal/activation peptides, the active chymases and intron regions, respectively. The lines attached to rectangles designate the 5' or Y nonceding regions. Arrows indicate the length and direction of sequences obtained. (B,C) The composite eDNA,' nucleotide and derived amino acid sequences for mouse ehymase 1 and 2, respectively. The numbers on the left indicate nucleotide positions. The amino acids deduced from the cDNA are shown in single-letter code above the first nucleotide of each codon; every tenth residue is numbered. Symbols used are: *, calalytic triad residues (His-45, Asp-89 and Ser-182); m, Asn-176 in the chymase I or Set-176 in the ch~mase 2 S~ binding pocket; *, potential N-linked glycosylation sites (Ash-59 in chymase 1; Asn-82 and Asn-100 in chymase 2); the putative polyadenylation signal sequences are underlined and the chymase 2 intron is double under lined.
85 M. C h y m . - 1 M. c h y m . - 2 MMCP-1 MMCP-2 MMCP-4 RMCP- I RMCP-II Dog C h y m . Chymotrypsin
MMLLTLHLLLLLLGSSTKAGE
IIGGTECIPH
SRPYMAYLEI
.... V . A R . . . . . . . . H.K. ..... V.AK .......... KF ..... V.SR ........ H... .... V . S R . . . . . . . . H... ..... V.S ......... H.D. ....... SK ........ H... IVNGEEAVPG SWPWQVSL--
MQAL.F.MA...P.GAG.E MQAL.F.MA...P.GAG.E MQAL.F.MA...P.GAG.E . . C . P . T M ..... C . R A E . E CGVPAIQPVLSGLSR
I
RMCP- I I Dog Chym. Chymotrypsin
LTAAHCAGRS
70
G .... A P Q . . G .... A P Q . • G .... T . Q . • G... VT.Q.. G .... S . Q . • G,.o.o°ooo GGSLINENWV
:
30
80
130
90
140
SFR-HNSQLC VGNPKKMQNV H Y K - D S L . I. . o S S T . V A S . HYD-Y.F.V° .°SST.LKTA DYD-YQL.V° A.S.TTLKSI HYD-Y.L.V. ..S.R.KRSA HYN-YNF.V. ..S.R. IRSA YYE-YKF.V. ..S.TTLRAA A.D-..L ....... R.TKSA GTKIKDAMIC AGAS--GVSS
I
190
I
i i
ILREN VIEGK VINGK .V.KSK VIKGE VIKGKD VIN V.KQ.KA
(226} (176 partial) (226) (224) (226} (227} (224) (228) (245)
QEPQACKHFT MGKK...I.K MDKE...MYK MDQE...DHN MDKE...NYW MDKE...NYF MDEK..VDYR M D .... R . Y H LSNTNCKKYW
160
150
200
110
DTLQEVKMRL R..R..EL.I E..R..EL.I V..R..EL.I ...R...L.I N..R...Q.I ¥..R..EL.I . . . . . . . L.. DRLQQASLPL
170
GIASY%q4RNR --KPPAVFTR ..V.SGRG.A --....I... ..V..GDSHG --. ....... ...S..-E .... A.A ..... .°V.°GRGDA --. . . . . . . . ..V.°GRGDA --. . . . . . . . ..V°.G.PDA --....I... . . V . . G Q N D A -- • . . . . . . . GIVSWGSSTC STSTPGVYAR
I
210 M. Chym.-1
M. C h y m . - 1 ISHYRPWINK M. C h y m . - 2 . . P H V .... R MMCP-I ..A.V...KT MMCP-2 ...Y.L...Y MMCP-4 . . S . V .... R RMCP-I ..P.V ..... RMCP-II V . T . V .... A Dog C h y m . . . . . . . . . . . Chymo~rypsinVTALVNWVQQTLAAN
50
I00
YKGDSGGPLL CAGIAQ---.M . . . . . . . . . . . V.H .... .M . . . . . . . . . . . V.H .... GQ ....... V .D.V.H .... ............. V.H .... ......... V ...V.H .... FM ........... V.H .... F ............ V.°---CMGDSGGPLV CKKNGAWTLV
i
180
L
40
KLTLGVGTLP LSANFNFIPP GRMCRAVGWG RTN-VNEPAS M..SA.DW. .P.PSD.AK. .T..W.A ..... G-LKKSI. E..PT.DVI. .PGPSD..D. .K..WTA... K.G-EK..T. E.NSD.DVIS .PSSSD..K..K..WTA... K.G-K.N.L. .E.PS.NVI..PRPSD..K..K .... A . . . . . G - . T . . T . .V.PR.DVI..PQPSD.LK..K .... A . . . Q . G - . T K . T . E..PA.NVV..PSPSD.-H..A..W.A... K.G-.RD.T. N.___~..R . . . . . . . P Q . . . v . . . . . . . V A . . . KRQ-o.G___SSG. SFSQTVSAVC LPSASDDFAA GTTCVTTGWG LTRYTNANTP 120
M. C h y m . - 1 M. C h y m . - 2 MMCP-I MMCP-2 MMCP-4 RMCP-I RMCP-II Dog Chym. Chymotrypsin
I.DRGSEDR. T.KN_~KER. T.ERGFTAT. T.ERG.KAT. ..EKGLRVI. L.LR.H.AS. -QDKTGFHFC
IMLLKLKEKA KTSKEDTWQK LEVEKQFLHP KYDENLWHD VRKR.C.Q.. IK...YI.P. N.NVSSKFN. .V .... E K Q . VSKS.S.Q.R I K .... I I . K N . N V S F N L Y . +++o+°E+.° INKN.P.Q.I IKT..T.V...FQYLSGFN ....... QK.. VSKT.S.Q.. I K .... I V . . . . N F Y S N L . . . . . . . . QK.. VSKT.S.Q.. I ..... IV.. N.NFYSNL ........ QK.. VRKR.S.Q.. I K .... I I . E S . N S V P N L . . . . . . . . EK.V IQK .......... I...P ..... DLTLR ............ QGSSSEKIQK LKIAKVFKNS KYNSLTINND ITLLKLSTAA
ITVLL-GAHN M..T.- .... ...T.-...D . S . I . - .... ...T.-...D T..T.-.V.D ...I.-...D .M.T.- .... SDVVVAGEFD
R ...... K..E M ..... R.SE M ..... S..E M ..... K..E ...... K..E ....... A.F VTAAHCGVTT 60
M. C h y m . - I M. C h y m . - 2 MMCP-1 MMCP-2 MMCP - 4 RMCP- I RMCP- I I Dog Chym. Chymotrypsin
SGFLIRRNFV
l
20 M. Chym.-1 M. Chym.-2 MMCP- 1 MMCP-2 MMCP-4 RMCP- I
VTSENYLSAC
l
--52% 54% 51% 59% 55% 60% 72% 34%
]
i
220 M.
230 chym.-2 52% 57% 68% 70% 69% 66% 54% 31%
240
Fig. 2. Primary structure alignments between mouse and other known chymases and bovine chymotrypsin. Residue ,umbering follows the standard chymotrypsinogen notation [21]. A single small dot indicates residues identical to those of mouse chymase I: gaps are indicated by dashes in the alignment. Important residues are noted as in Fig. t. The vertical arrow (1') marks the predicted signal peptida~ cleavage site in mouse chymase 1. The number of residues is listed in parentheses at the COOH end of each enzyme. Identity percentages relative to each mouse chymase are given. The sources of the sequence data are as follow: mouse chymases (M. Chym., this study), MMCP-1 [3], MMCP-2 [41, MMCP-4 [5], RMCP 1 [20], RMCP II [7], dog chymase (Dog. Chym., 15) and bovine chymotrypsin [211.
MMCP-5 [1] indicate that it may be analogous to mouse chymase 1. Of the seven cysteine residues, six occur at identical positions in other chymases and chymotrypsin. These cysteines may form three disulfide bridges as in chymotrypsin, suggesting a conserved tertiary structure among this enzyme superfamily. A potential N-linked glycosylation site is present at Ash72 (Ash-59 in Fig. IB). Based on the number of basic (Arg + Lys = 30) and acidic (Asp + Glu = 17) amino acids, the mature enzyme is positively charged at pH 7.4. This high positive charge ( + 13) distinguishes chy-
mase 1 from dog chymase ( + 7); among chymases only MMCP-4 ( + 15 [5]) and mouse chymase 2 ( + 17, below) are so positively charged. Another interesting feature of mouse chymase 1 is the presence of Ash-189 rather than Ser in the normal S~ substrate-binding site of chymotryptic enzymes with substrate preferences for hydrophobic amino acids. Although Thr and Ala were found in the corresponding position of MMCP-1 and RMCP-II, respectively, Asn has not been identified in any other serine proteinase. The binding site Asn may confer on chymase I
86 a preference for polar or charged residues rather than the neutral, hydrophobic residue specificity normally observed for chymotrypsin-like enzymes. These data suggest that the mast cell chymases may vary considerably with regard to substrate specificity. The complete leader peptide for chymase 1 is identical in length to that of dog chymase, but one amino acid longer than that of MMCP-2, MMCP-4 and RMCP II. However, the mouse and dog chymase leader peptides differ significantly in content of charged amino acids: the leader of mouse chymase 1 is uncharged overall, while in dog it bears a net positive charge ( + 8). Based on the ' - 3 , - 1 ' rule of yon Heijne [15], the predicted signal peptidase cleavage leaves the two amino acid 'Pro' peptide Gly-Glu (Fig. 2). In contrast, similar processing of other chymase preproproteins would leave Glu-Glu propeptides. This scheme suggests an activation process in common with the granule-associated serine proteinases human cathepsin G and neutrophil elastase [16]. A partial eDNA of 901 bases for a second chymase was obtained having an ORF of 535 bases flanked by 5' and 3' untranslated regions of 116 and 250 bases, respectively (Fig. 1A and C). The 5' noncoding sequence may represent a residual intron at the highly conserved intron/exon junction at this location in the sedne proteinase superfamily [7]. The 177 amino acids of the carboxy terminus of chymase 2 are 66-70% identical to the same regions of MMCP-2, MMCP-4, RMCP I and RMCP II (Fig. 2). Based on the abundance of basic over acidic residues (28 vs. 11), this large (~71%) carboxy terminal portion of chymase 2 is positively charged (+ 17) at physiological pH and most similar to MMCP-4 (+ 19 over the analogous carboxy terminal region). Chymase 2, like other chymotrypsinlike proteinases, contains a Ser-189 in its S t substratebinding pocket. It also contains a second potential glycosylation site at Asn-ll3, found elsewhere only in the dog chymase. Northern blot analyses were performed with mouse chymase cDNA probes to determine their tissuespecific expression. In addition, the blot was probed with tubulin cDNA to measure mRNA integrity and housekeeping gene expression. In contrast to tubulin, significant amounts of chymase 1 transcripts were observed only in the ABFTL-6 mRNA (Fig. 3A and B). Levels of chymase 2 transcripts detected in tongue, intestine and ABFTL-6 cell RNAs (Fig. 3C) are distinctly different from those found for mouse chymase 1. Strong chymase 2 signals were obtained with both the ABFTL-6 and tongue RNAs, with low, but detectable levels in intestine RNA. Tongue has been used for mast cell chymase isolation [17] and the intense hybridization observed to tongue RNA indicates that chymase 2 expression in tongue is remarkably high. This contrasts with the levels of chymase 1 (Fig.
A kb
a
b
B c
a
b
C c
a
b
c
i
1.10.6Fig. 3. Northern-blot hybridization analysis for chymase expression. Poly(A) + RNA from mouse tongue (6 pg, lane a). intestine (6 pg, lane b) and ABFTL-6 cells (2.4/zg, lane c) were fractionated on a formaldehyde-denaturing 1.5% agarose gel and blotted onto nitrocellulose. The blot was sequentially hybridized with cDNAs for chicken ot-tubulin (panel A), mouse chymase I (panel B) and mouse chymase 2 (panel C). M r markers are on the left in kilobases (kb).
3B) and tryptase transcripts which are much higher in ABFTL-6 mRNA than in tongue or intestine mRNA [18]. Regarding the expression of other chymases, MMCP-1 and MMCP-2 are selectively expressed in different mucosal mast cell populations [4,19], whereas MMCP-4 is expressed in both connective tissue and mucosal mast cells [5]. In addition, MMCP-3 and MMCP-5 were identified in serosal connective tissue mast cells [1]. Chymases 1 and 2 are distinct from the three previously described mouse chymases (MMCP-I, -2 and -4). These differences occur in the mRNA and amino acid sequences, and include presumed substrate specificities and potential protein glycosylations. Thus, these results provide additional evidence that in mice the relative amounts of each proteinase gene transcript depends on mast cell location and type. Acknowledgments This work was supported by Grants GM41952 (to PRM) and HIA2623 (to DAJ) from the National Institutes of Health. We would like to thank Dr. J. Pierce (National Institutes of Health) for the ABFTL-6 cells, Dr. P. Leder (Harvard University) for RMCP II eDNA, Dr. C. Smith (Promega) for mouse tissue RNAs and Ms. Melissa Phillips for technical assistance. References 1 Reynolds, D.S., Stevens, R.L, Lane, W.S. and Carl-, M.H. (1990) Proc. Natl. Acad. Sci. USA 87, 3230-3234. 2 Newlands, G.F.J., Gibson, S., Knox, D.P., Grencis, R., Wakelin, D. and Miller, H.R.P. (1987) Immunology 62, 629-634. 3 Le Trong, H., Newlands, G.F.J., Miller, H.R.P., Charbonneau, H., Neurath, H. and Woodbury, R.G. (1989) Biochemistry 28, 391-395.
87 4 Serafin, W.E., Reynolds, D.S., Rogel J.S., Lane, W.S., Conder, G.A., Johnson, S.S., Austen, K.F. and Stevens, R.L. (1990) J. Biol. Chem. 265, 423-429. 5 Serafin, W.E., Sullivan, T.P., Conder, G.A., Ebrahimi, A., Marchain, P., Johnson, S.S., Austen, K.F. and Reynolds, D.S. (1,°91) J. Biol. Chem. 266, 1934-.1941. 6 Pierce, J.H., Di Fiore, P.P., Aaronson, S.A., Polter, M., Pumphrey, J., Scott, A. and lhle, J.N. (1985) Cell 41,685-693. 7 Benfe3,, P.N., Yin, F.H. and Leder, P. (1987)J. Biol. Chem. 262, 5377-5384. 8 Chirgwin, J.M., Prztd~a, A.E., MacDonald, RJ. and Rutter, WJ. (1979) Biochemistry 18, 5294-5299. 9 Aviv, H. and Leder, P. (1972) Ptoc. Natl. Acad. Sci. USA 69, 1408-1412. 10 Musich, P.R. and Dykes, R. (1986) Proc. Natl. Acad. Sci. USA 83, 4854-4858. 11 McConaughy, B,L., Laird, C.D. and McCarthy, BJ. (1969) Biochemistry 8, 3289-3295.
12 Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 13 Verma, M. (1989) BioTechniques 7, 230-232. 14 Caughey, G.H., Raymond, W.W. and Vanderslice, P. (1990) Biochemistry 29, 5166-5171. 15 Von Heijne, (3. (1986) Nucleic Acid Res. 14, 4683-4690. 16 Salvesen, G. and Enghild, J. (I990) Biochemistry 29, 5304-5308. 17 Kido, H., Fukusen, N. and Katunuma, N. 0984) Anal. Biochem. 137, 449-453. 18 Chu, W. (1991) Dissertation, East Tenn. State University. 19 Miller, H.R.P., Huntley, J.F., Newlands, G.FJ., Mackellar, A., Laminas, D.A- and Wakelin, D. (1988) Immunology 65, 559-566. 20 Le Trong, H., Parm.elee, D.C., WaIsh, K.A., Neurath, H. and Woodbury, R.C. (1987) Biochemistry 26, 6988-6994. 2I Blow, D.M., Biektoft, I J . and Hartley, B.S. (1969) Nature 221, 337-340.
83
BBAPRO 34194
Molecular cloning and characterization of mouse mast cell chymases Wei Chu 1, David A. Johnson and Phillip R. Musich Department of Biochemistry, James H. QuiUenCollegeof Medicine, East TennesseeState Unicersity,Johnson City, TN (USA)
(Received 7 August 1991) (Revised manuscript received 11 December 1991)
Key words: cDNA; Mast cell; Proteinase; Amino acid seq,-.nce
Mouse mast cell chymases are granule-associated serine proteinases with chymotrypsin-like substrate specificities, cDNAs for two new chymases were isolated from a cDNA library constructed using mRNA from ABFTL-6 mouse mast cells by screening with a rat mast cell proteinase cDNA. The deduced amino acid sequence of mouse chymase 1 consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. Chymase I is unusual in that an Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine proteinase. Also, chymase 1 is expected to have a large positive charge ( + 13) at physiological pH. A partial cDNA for chymase 2 encodes 177 residues of the carboxy terminal portion of a second proteinase distinct from chymase I. Chymase 2 cDNA contains a highly conserved intron/exon junction, a high positive charge ( + 17) and a novel, second potential N-glycosylation site. Tt,mscripts for both chymases are found in ABFTL-6 mast cells, but only chymase 2 mRNA is in mouse connectk,e tissue mast cells. These data suggest that these chymases have distinct enzymatic properties and tissue-specific patterns of gene expression.
Introduction Mast cells contain granule-associated proteolytic enzymes with both trypsin-like and chymotrypsin-like specificities, referred to as tryptase and chymase, respectively. In the mouse, six distinct chymotryptic proteinases (denoted as MMCP-I through MMCP-6) have been identified in mast cells based on partial sequencing of their amino termini [1]. Complete sequences for several of these chymases have been determined either from the purified proteins or based on the nucleotide sequences of their cloned cDNAs. The first chymase, mouse mast cell proteinase (MMCP)-I, was isolated from mice infected with Trichinefla spiralis [2] and its amino acid sequence was determined [3]. Subsequently, a MMCP-2 cDNA was identified from Kirsten sarcoma virus-immortalized mast cells [4]. Recently, sequences
t Present address: Molecular Genetics Department, Hoffman-La Roche, Nutley, N J, USA. Abbreviations: cDNA, complementary DNA; kb, kilobascs; MMCP, mouse mast cell proteinase; ORF, open reading frame; RMCP, rat mast cell pmteinase. This article has been submitted to GENBANK; accession numbers: chymase 1, m68898; chymase 2, M68899. Correspondence: P.R. Musich, Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0581, USA.
for chymase MMCP-4 have been reported [5]. ABFTL-6 mouse mast cells, an Abelson virus-transformed line [6], contain R N A homologous to rat mast cell proteinase (RMCP) II cDNA [7]. Thus, the chymotryptic composition of mouse mast cells may depend on the specific mast cell type, its tissue micro-environment and its state of activation. This report describes the cloning, characterization and transcriptional analysis of complementary DNAs (cDNAs) encoding mouse mast cell chymases different from those previously reported. The deduced amino acid sequences of these chymases indicate novel substrate preferences, ionic interactions and secondary modifications which distinguish them from other, known chyrnases.
Materials and Methods R N A isolation and cDNA library construction. ABFFL-6 mast cells were grown as described [6] and their R N A isolated by the guanidine thiocyanate/CsCI density gradient centrifugation method [8]. Poly(A) + RNA was purified by two cycles of chromatography on oligo(dT)-cellulose [9]. The c D N A library was prepared using a Pharmacia cDNA synthesis kit and Stratagene Agtl0 cloning and Gigapack packaging kits. Positive clones were identified by hybridization screening of
84 plaque lifts of the unamplified library with RMCP II eDNA probe to isolate clones AC1 through AC9. The library was rescreened with the insert of the recombinant ,~C3 identified in the first round to isolate additional positive clones. Hybridizations were performed under standard conditions [10] at 42°C in a buffer containing 0.45 M NaCI, 0.045 M sodium citrate, 10 mM Tris-HCI (pH 7.4), 0.2% sodium dodecyl sulfate, 2% non-fat dry milk and 50% formamide, followed by washes at 65°C in 0.45 M NaCI-0.045 M sodium citrate containing 0.1% sodium dodecyl sulfate. These conditions require the stable hybrids to have 86% sequence identity to mouse chymase eDNA (49% G + C [11]). DNA probes were labeled with [32p]dATP by the random primer method [12]. Isolation and sequencing of recombinants. DNA from plaque-purified phage was isolated [13] and the cDNA inserts and their fragments subcloned into pTZI9U plasmid for sequence analysis by the dideoxy chain termination method using the Sequenase TM protocol (United States Biochemicals). Data were analyzed using Hitachi DNASIS/PROSIS software.
Mouse Chymue 2
Mouse ChymMe 1
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14 chymase clones were selected for sequence analysis. The nucleotide sequence for chymase 1, compiled from clones p,~C19, 26 and 51.1, and its derived amino acid sequence are shown in Fig. 1A and B. The cDNA is 960 bp long and contains a 744 bp open reading frame (ORF) adjacent to 5' and 3' untranslated regions of 5 and 211 bases, respectively. The translated sequence consists of 247 amino acids including a hydrophobic signal peptide of 21 amino acids. Mature mouse chymase 1 contains 226 amino acids with a calculated M r of 25.4 kDa, equal to that of dog mast cell chymase [14], MMCP-I [3] and MMCP-4 [5], but two amino acids longer than MMCP-2 [4]. Mouse chymase 1 exhibits many features characteristic of serine proteinases (Fig. 2), including the highly conserved residues comprising the catalytic 'charge-relay' system corresponding to the His-57, Asp-102 and Set-195 in chymotrypsin. Its sequence identity ranges from 72% with dog chymase to 34% with bovine chymotrypsin. The amino-terminal sequence data of
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Results and Discussion
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-i *I A G E I T ~ AG AG AT
?1
10 20 I G G T E C l P H S R P Y M A ¥ L E I V T S E CATTGGAGGCACGGAG TGCATACCACAC'1'CCCGCCCCTACATGGCCTATCTGGAAATTGTC &CTT ~ G A G
141
30 40 N Y U S A C S G • L ~ R R N F V L T & A H ~---JL~C C~GT CGG¢C'IGP . J ~ . G C ] ~ CCTGATRJ~, J ~ G ~ G W I " G CIGA CTGC~ ~ ~
211
50 * 60 70 R S Z T V L L G & H N K T S K S D T W Q K r_ E G~ G G T C C A T ~ C A 6 T C C I C C I ~ k G G A G C C C A T ~ C ~ C J 4 T CTRA~GJUtGACACGT~CAG ~ G ~ G A
281
80 • 90 V E R Q F L H P K • D E N L V V H D i M L GGTGGRAkI~c ~A'rCCCI'TCATCCItA,kATATG&TG WJ &I~TTTGGTTGTCCACGAC~T CATG~
351
1O0 110 L X E K A K L T L G V G T L P L S A N F N l:' I P ~ G A~G G&G.MIUIK;CCiM~GC~AACCC~AGGTG~X~GG~ACCCCCCC~CTCTCTGCC~ c ~ r CAN: ~ TR TC C
421
120 130 140 P G R Iq C R & V O W G R T N V N E P & S D T L C&CCCGGGAG~J4TGTGC ~G(;CAGTTGGC1T~.GG CAGAACk~.CG ¢GPJ~TGAGCCAGC~ ~ G A C A C A ~
491
150 160 Q it Y R M R L Q E P '~ A C K H F T S F R H N S ~GT;tAN;&~;N;&CTCCAGGRG L'CCCRd~ CCI~GC~CRCI~CACCRGT ~ T C ~ C A C ~ C C
561
170 i 180 • 9 L c V G el IP K K M Q N v Y K G D s G G P ~ CAOCT'GTGTGTGGGCAACCCC,g,&OF.AOA'J~CAAAAI'GTATACAA(~GAG&CTCTGGAGGRC~~
fi31
190 200 210 A G X A Q G X A S Y q H R N M K P P A V F T R GTG~I"RGC~R~,C&T~GCAT¢CZWrGTACATCGGR.R'~CG&RAGCCCCCTGCTGT~ ~ R C ~ G
701 771 841 911
22O I S H • R P W I N It I L R E H End RRTCTCC~TTAC~C CeT~TCR~TRAGATCTTGA~OAATTARCT~G GAG ~ G C ~ GTGAGGAAATCTGGRACTGGRJ4T>GCAGGTTTTGTGTG CC&TGCG&TCTGGC ~ T ~ G T ~ C ~ C1~GARG4:CCIX;CC~GGTCC CeGRGC~CCAGRAGG~r C T e A C P I ~ G T C A C A G ~ A ~ C ~ C ~ C C ATCTTC~TT J ~ C C T ~ G & ~ T T GTCA.q.CTGC~ 960
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141
60 70 80 H N V R K R E C T Q ~ K I K V E K 1" I L, P P N TCAT~TGTGAGAAAGAGAGARTGCACA~ CAGAJ~GATRAR~GTI"GRJM~GTACATCTTGCCTCC~T
211
. • 90 100 Y N V S S K F N D I V L L K L E K Q & M,~ L T $ A TAC.ARTGTGTC~C~GTTCARI~I;RCATCGTA~Z'ACTGRA~"]'GAP.ARGCARGCTJ~"n'GA~
28]
]10 120 V D V V P L P A P S D F A K E> G T M C W A & G CTGTGGATG TAGT TCCCCTGCC~GCCCC CTCTG&CT~r~G;CCRJ4GCC~GGGACGATGCGC ' J ~ , G G C A G ~
35]
130 W G R T G L K X S CTG GGC,G CG R A C T G G A T T G A ~ T
421
K K ~ G G
491
A V Y ~ G D G G P L L C A G V R N G I V $ TGG.CRTCAGTAT~C ATGGGAGRCTCTGGAGGACCTCTR.CTGTGTGC T C . G G G T G G C C C , ~ . ~ T A ~ T A ~
561
200 210 220 S G R G N A K P P A I F T R I S P H V P kl I N TT CTGGACGCGGRJ~ATGCF_%~GCCCCCTGCRJ~'~J~CRC C C C ~ T C T C C C C A C & T G ~ ; C C C I G G & ~ T ~
631 701 771 841
226 R V l E G K End AGAGTCA'~AGAGGGCAAGTAGTGAAR~GCCTGACCTGCGTGCATCAGAGTCTTCAAGCCAGAGCTCTT~ G & T A A C C C T T G G G T TC RR C A J ~ A T G T G T CCAT C C ~ T CCCCTGCCTGC(~CC~A~?J%TG T C C t ' T ~ A T G T C C C C C G C C TG CC C T C A C ~ G C C C C C A G T C TGTCCCCRJ%GATG&~TAJ4AGTCTGT~ &TGA T G G A C C G T T C C C T G T ~ 901
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150 170 C K I F K H Y K D $ I. ~ I C V G S S C C T G T A ~ J % A T A T T T A R G C & T T R C R A G G A T A G C C T C C ~ G AT C T G ~ G ~ G T T
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Fig. 1. (A) Subcloning and sequencing strategy for mouse chymase 1 and 2 cDNAs. The rectangles repre~nts ORFs. The speckled, open and shaded areas repre~nt the putative signal/activation peptides, the active chymases and intron regions, respectively. The lines attached to rectangles designate the 5' or Y nonceding regions. Arrows indicate the length and direction of sequences obtained. (B,C) The composite eDNA,' nucleotide and derived amino acid sequences for mouse ehymase 1 and 2, respectively. The numbers on the left indicate nucleotide positions. The amino acids deduced from the cDNA are shown in single-letter code above the first nucleotide of each codon; every tenth residue is numbered. Symbols used are: *, calalytic triad residues (His-45, Asp-89 and Ser-182); m, Asn-176 in the chymase I or Set-176 in the ch~mase 2 S~ binding pocket; *, potential N-linked glycosylation sites (Ash-59 in chymase 1; Asn-82 and Asn-100 in chymase 2); the putative polyadenylation signal sequences are underlined and the chymase 2 intron is double under lined.
85 M. C h y m . - 1 M. c h y m . - 2 MMCP-1 MMCP-2 MMCP-4 RMCP- I RMCP-II Dog C h y m . Chymotrypsin
MMLLTLHLLLLLLGSSTKAGE
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SRPYMAYLEI
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MQAL.F.MA...P.GAG.E MQAL.F.MA...P.GAG.E MQAL.F.MA...P.GAG.E . . C . P . T M ..... C . R A E . E CGVPAIQPVLSGLSR
I
RMCP- I I Dog Chym. Chymotrypsin
LTAAHCAGRS
70
G .... A P Q . . G .... A P Q . • G .... T . Q . • G... VT.Q.. G .... S . Q . • G,.o.o°ooo GGSLINENWV
:
30
80
130
90
140
SFR-HNSQLC VGNPKKMQNV H Y K - D S L . I. . o S S T . V A S . HYD-Y.F.V° .°SST.LKTA DYD-YQL.V° A.S.TTLKSI HYD-Y.L.V. ..S.R.KRSA HYN-YNF.V. ..S.R. IRSA YYE-YKF.V. ..S.TTLRAA A.D-..L ....... R.TKSA GTKIKDAMIC AGAS--GVSS
I
190
I
i i
ILREN VIEGK VINGK .V.KSK VIKGE VIKGKD VIN V.KQ.KA
(226} (176 partial) (226) (224) (226} (227} (224) (228) (245)
QEPQACKHFT MGKK...I.K MDKE...MYK MDQE...DHN MDKE...NYW MDKE...NYF MDEK..VDYR M D .... R . Y H LSNTNCKKYW
160
150
200
110
DTLQEVKMRL R..R..EL.I E..R..EL.I V..R..EL.I ...R...L.I N..R...Q.I ¥..R..EL.I . . . . . . . L.. DRLQQASLPL
170
GIASY%q4RNR --KPPAVFTR ..V.SGRG.A --....I... ..V..GDSHG --. ....... ...S..-E .... A.A ..... .°V.°GRGDA --. . . . . . . . ..V.°GRGDA --. . . . . . . . ..V°.G.PDA --....I... . . V . . G Q N D A -- • . . . . . . . GIVSWGSSTC STSTPGVYAR
I
210 M. Chym.-1
M. C h y m . - 1 ISHYRPWINK M. C h y m . - 2 . . P H V .... R MMCP-I ..A.V...KT MMCP-2 ...Y.L...Y MMCP-4 . . S . V .... R RMCP-I ..P.V ..... RMCP-II V . T . V .... A Dog C h y m . . . . . . . . . . . Chymo~rypsinVTALVNWVQQTLAAN
50
I00
YKGDSGGPLL CAGIAQ---.M . . . . . . . . . . . V.H .... .M . . . . . . . . . . . V.H .... GQ ....... V .D.V.H .... ............. V.H .... ......... V ...V.H .... FM ........... V.H .... F ............ V.°---CMGDSGGPLV CKKNGAWTLV
i
180
L
40
KLTLGVGTLP LSANFNFIPP GRMCRAVGWG RTN-VNEPAS M..SA.DW. .P.PSD.AK. .T..W.A ..... G-LKKSI. E..PT.DVI. .PGPSD..D. .K..WTA... K.G-EK..T. E.NSD.DVIS .PSSSD..K..K..WTA... K.G-K.N.L. .E.PS.NVI..PRPSD..K..K .... A . . . . . G - . T . . T . .V.PR.DVI..PQPSD.LK..K .... A . . . Q . G - . T K . T . E..PA.NVV..PSPSD.-H..A..W.A... K.G-.RD.T. N.___~..R . . . . . . . P Q . . . v . . . . . . . V A . . . KRQ-o.G___SSG. SFSQTVSAVC LPSASDDFAA GTTCVTTGWG LTRYTNANTP 120
M. C h y m . - 1 M. C h y m . - 2 MMCP-I MMCP-2 MMCP-4 RMCP-I RMCP-II Dog Chym. Chymotrypsin
I.DRGSEDR. T.KN_~KER. T.ERGFTAT. T.ERG.KAT. ..EKGLRVI. L.LR.H.AS. -QDKTGFHFC
IMLLKLKEKA KTSKEDTWQK LEVEKQFLHP KYDENLWHD VRKR.C.Q.. IK...YI.P. N.NVSSKFN. .V .... E K Q . VSKS.S.Q.R I K .... I I . K N . N V S F N L Y . +++o+°E+.° INKN.P.Q.I IKT..T.V...FQYLSGFN ....... QK.. VSKT.S.Q.. I K .... I V . . . . N F Y S N L . . . . . . . . QK.. VSKT.S.Q.. I ..... IV.. N.NFYSNL ........ QK.. VRKR.S.Q.. I K .... I I . E S . N S V P N L . . . . . . . . EK.V IQK .......... I...P ..... DLTLR ............ QGSSSEKIQK LKIAKVFKNS KYNSLTINND ITLLKLSTAA
ITVLL-GAHN M..T.- .... ...T.-...D . S . I . - .... ...T.-...D T..T.-.V.D ...I.-...D .M.T.- .... SDVVVAGEFD
R ...... K..E M ..... R.SE M ..... S..E M ..... K..E ...... K..E ....... A.F VTAAHCGVTT 60
M. C h y m . - I M. C h y m . - 2 MMCP-1 MMCP-2 MMCP - 4 RMCP- I RMCP- I I Dog Chym. Chymotrypsin
SGFLIRRNFV
l
20 M. Chym.-1 M. Chym.-2 MMCP- 1 MMCP-2 MMCP-4 RMCP- I
VTSENYLSAC
l
--52% 54% 51% 59% 55% 60% 72% 34%
]
i
220 M.
230 chym.-2 52% 57% 68% 70% 69% 66% 54% 31%
240
Fig. 2. Primary structure alignments between mouse and other known chymases and bovine chymotrypsin. Residue ,umbering follows the standard chymotrypsinogen notation [21]. A single small dot indicates residues identical to those of mouse chymase I: gaps are indicated by dashes in the alignment. Important residues are noted as in Fig. t. The vertical arrow (1') marks the predicted signal peptida~ cleavage site in mouse chymase 1. The number of residues is listed in parentheses at the COOH end of each enzyme. Identity percentages relative to each mouse chymase are given. The sources of the sequence data are as follow: mouse chymases (M. Chym., this study), MMCP-1 [3], MMCP-2 [41, MMCP-4 [5], RMCP 1 [20], RMCP II [7], dog chymase (Dog. Chym., 15) and bovine chymotrypsin [211.
MMCP-5 [1] indicate that it may be analogous to mouse chymase 1. Of the seven cysteine residues, six occur at identical positions in other chymases and chymotrypsin. These cysteines may form three disulfide bridges as in chymotrypsin, suggesting a conserved tertiary structure among this enzyme superfamily. A potential N-linked glycosylation site is present at Ash72 (Ash-59 in Fig. IB). Based on the number of basic (Arg + Lys = 30) and acidic (Asp + Glu = 17) amino acids, the mature enzyme is positively charged at pH 7.4. This high positive charge ( + 13) distinguishes chy-
mase 1 from dog chymase ( + 7); among chymases only MMCP-4 ( + 15 [5]) and mouse chymase 2 ( + 17, below) are so positively charged. Another interesting feature of mouse chymase 1 is the presence of Ash-189 rather than Ser in the normal S~ substrate-binding site of chymotryptic enzymes with substrate preferences for hydrophobic amino acids. Although Thr and Ala were found in the corresponding position of MMCP-1 and RMCP-II, respectively, Asn has not been identified in any other serine proteinase. The binding site Asn may confer on chymase I
86 a preference for polar or charged residues rather than the neutral, hydrophobic residue specificity normally observed for chymotrypsin-like enzymes. These data suggest that the mast cell chymases may vary considerably with regard to substrate specificity. The complete leader peptide for chymase 1 is identical in length to that of dog chymase, but one amino acid longer than that of MMCP-2, MMCP-4 and RMCP II. However, the mouse and dog chymase leader peptides differ significantly in content of charged amino acids: the leader of mouse chymase 1 is uncharged overall, while in dog it bears a net positive charge ( + 8). Based on the ' - 3 , - 1 ' rule of yon Heijne [15], the predicted signal peptidase cleavage leaves the two amino acid 'Pro' peptide Gly-Glu (Fig. 2). In contrast, similar processing of other chymase preproproteins would leave Glu-Glu propeptides. This scheme suggests an activation process in common with the granule-associated serine proteinases human cathepsin G and neutrophil elastase [16]. A partial eDNA of 901 bases for a second chymase was obtained having an ORF of 535 bases flanked by 5' and 3' untranslated regions of 116 and 250 bases, respectively (Fig. 1A and C). The 5' noncoding sequence may represent a residual intron at the highly conserved intron/exon junction at this location in the sedne proteinase superfamily [7]. The 177 amino acids of the carboxy terminus of chymase 2 are 66-70% identical to the same regions of MMCP-2, MMCP-4, RMCP I and RMCP II (Fig. 2). Based on the abundance of basic over acidic residues (28 vs. 11), this large (~71%) carboxy terminal portion of chymase 2 is positively charged (+ 17) at physiological pH and most similar to MMCP-4 (+ 19 over the analogous carboxy terminal region). Chymase 2, like other chymotrypsinlike proteinases, contains a Ser-189 in its S t substratebinding pocket. It also contains a second potential glycosylation site at Asn-ll3, found elsewhere only in the dog chymase. Northern blot analyses were performed with mouse chymase cDNA probes to determine their tissuespecific expression. In addition, the blot was probed with tubulin cDNA to measure mRNA integrity and housekeeping gene expression. In contrast to tubulin, significant amounts of chymase 1 transcripts were observed only in the ABFTL-6 mRNA (Fig. 3A and B). Levels of chymase 2 transcripts detected in tongue, intestine and ABFTL-6 cell RNAs (Fig. 3C) are distinctly different from those found for mouse chymase 1. Strong chymase 2 signals were obtained with both the ABFTL-6 and tongue RNAs, with low, but detectable levels in intestine RNA. Tongue has been used for mast cell chymase isolation [17] and the intense hybridization observed to tongue RNA indicates that chymase 2 expression in tongue is remarkably high. This contrasts with the levels of chymase 1 (Fig.
A kb
a
b
B c
a
b
C c
a
b
c
i
1.10.6Fig. 3. Northern-blot hybridization analysis for chymase expression. Poly(A) + RNA from mouse tongue (6 pg, lane a). intestine (6 pg, lane b) and ABFTL-6 cells (2.4/zg, lane c) were fractionated on a formaldehyde-denaturing 1.5% agarose gel and blotted onto nitrocellulose. The blot was sequentially hybridized with cDNAs for chicken ot-tubulin (panel A), mouse chymase I (panel B) and mouse chymase 2 (panel C). M r markers are on the left in kilobases (kb).
3B) and tryptase transcripts which are much higher in ABFTL-6 mRNA than in tongue or intestine mRNA [18]. Regarding the expression of other chymases, MMCP-1 and MMCP-2 are selectively expressed in different mucosal mast cell populations [4,19], whereas MMCP-4 is expressed in both connective tissue and mucosal mast cells [5]. In addition, MMCP-3 and MMCP-5 were identified in serosal connective tissue mast cells [1]. Chymases 1 and 2 are distinct from the three previously described mouse chymases (MMCP-I, -2 and -4). These differences occur in the mRNA and amino acid sequences, and include presumed substrate specificities and potential protein glycosylations. Thus, these results provide additional evidence that in mice the relative amounts of each proteinase gene transcript depends on mast cell location and type. Acknowledgments This work was supported by Grants GM41952 (to PRM) and HIA2623 (to DAJ) from the National Institutes of Health. We would like to thank Dr. J. Pierce (National Institutes of Health) for the ABFTL-6 cells, Dr. P. Leder (Harvard University) for RMCP II eDNA, Dr. C. Smith (Promega) for mouse tissue RNAs and Ms. Melissa Phillips for technical assistance. References 1 Reynolds, D.S., Stevens, R.L, Lane, W.S. and Carl-, M.H. (1990) Proc. Natl. Acad. Sci. USA 87, 3230-3234. 2 Newlands, G.F.J., Gibson, S., Knox, D.P., Grencis, R., Wakelin, D. and Miller, H.R.P. (1987) Immunology 62, 629-634. 3 Le Trong, H., Newlands, G.F.J., Miller, H.R.P., Charbonneau, H., Neurath, H. and Woodbury, R.G. (1989) Biochemistry 28, 391-395.
87 4 Serafin, W.E., Reynolds, D.S., Rogel J.S., Lane, W.S., Conder, G.A., Johnson, S.S., Austen, K.F. and Stevens, R.L. (1990) J. Biol. Chem. 265, 423-429. 5 Serafin, W.E., Sullivan, T.P., Conder, G.A., Ebrahimi, A., Marchain, P., Johnson, S.S., Austen, K.F. and Reynolds, D.S. (1,°91) J. Biol. Chem. 266, 1934-.1941. 6 Pierce, J.H., Di Fiore, P.P., Aaronson, S.A., Polter, M., Pumphrey, J., Scott, A. and lhle, J.N. (1985) Cell 41,685-693. 7 Benfe3,, P.N., Yin, F.H. and Leder, P. (1987)J. Biol. Chem. 262, 5377-5384. 8 Chirgwin, J.M., Prztd~a, A.E., MacDonald, RJ. and Rutter, WJ. (1979) Biochemistry 18, 5294-5299. 9 Aviv, H. and Leder, P. (1972) Ptoc. Natl. Acad. Sci. USA 69, 1408-1412. 10 Musich, P.R. and Dykes, R. (1986) Proc. Natl. Acad. Sci. USA 83, 4854-4858. 11 McConaughy, B,L., Laird, C.D. and McCarthy, BJ. (1969) Biochemistry 8, 3289-3295.
12 Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 13 Verma, M. (1989) BioTechniques 7, 230-232. 14 Caughey, G.H., Raymond, W.W. and Vanderslice, P. (1990) Biochemistry 29, 5166-5171. 15 Von Heijne, (3. (1986) Nucleic Acid Res. 14, 4683-4690. 16 Salvesen, G. and Enghild, J. (I990) Biochemistry 29, 5304-5308. 17 Kido, H., Fukusen, N. and Katunuma, N. 0984) Anal. Biochem. 137, 449-453. 18 Chu, W. (1991) Dissertation, East Tenn. State University. 19 Miller, H.R.P., Huntley, J.F., Newlands, G.FJ., Mackellar, A., Laminas, D.A- and Wakelin, D. (1988) Immunology 65, 559-566. 20 Le Trong, H., Parm.elee, D.C., WaIsh, K.A., Neurath, H. and Woodbury, R.C. (1987) Biochemistry 26, 6988-6994. 2I Blow, D.M., Biektoft, I J . and Hartley, B.S. (1969) Nature 221, 337-340.
