Cloning and sequencing of mouse VCAM-1 cDNA

Gene, 126 (1993) 261-264 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00

261

GENE 0705 1

Cloning and sequencing of mouse VCAM-1 cDNA (Endothelial cells; adhesion molecules; immunoglobulin

gene superfamily; amino acid sequence homology)

Masatake Araki, Kimi Araki and Pierre Vassalli Department of Pathology, C.M.U.,

University of Geneva, CH-1211 Geneva, Switzerland

Received by J.K.C. Knowles: 29 October 1992; Revised/Accepted: 21 December/22 December 1992; Received at publishers: 26 January 1993

SUMMARY

We report here the nucleotide sequence of a cDNA encoding the mouse vascular cell adhesion molecule-l (VCAMl), a member of the immunoglobulin gene superfamily. The 2972-bp cDNA encodes 739 amino acids (aa) and shows 76.4% and 75.9% similarity with the human VCAM-1 nucleotide (nt) and aa sequence, respectively. Three independent cDNA clones had a 7-Ig unit structure (molecule containing a seven ‘immunoglobulin motif’ repeat), and only sequences corresponding to the 7- (and not to the 6)-Ig unit VCAM were detected by reverse transcription polymerase chain reaction (RT-PCR), indicating that in the mouse this is the dominant, if not exclusive, form of this molecule. Unlike other adhesion molecules, the cytoplasmic tails (19 aa) of mouse and human VCAM-1 are identical, despite a 85.2% similarity only at the nt level, suggesting a critical functional role.

INTRODUCTION

Vascular cell adhesion molecule-l (VCAM-1) was identified on activated HUVECs by the monoclonal antibody El/6 (Rice and Bevilacqua, 1989) and by expression cloning (Osborn et al., 1989). This surface molecule is not constitutively expressed on endothelial cells and is induced in vitro by exposure to the cytokine TNF and IL-l; it has been shown to mediate intercellular adhesion via interaction with a counterreceptor, the integrin ‘very late antigen-Q (VLA4), which is expressed on monocytes, lymphocytes, basophils, eosinophils, and certain tumor cells, but not neutrophils (Elites et al., 1990; Rice et al., Correspondence to: Dr. P. Vassalli, Department of Pathology, Centre Medical Universitaire, 1, rue Michel-Servet, CH-1211 Geneve 4, Switzerland. Tel. (022) 702-5747; Fax (022) 347-3334.

Abbreviations: aa, amino acid(s); bp, base pair(s); CAM, cell adhesion molecule; CP, cytoplasmic tail; h, human; HUVECs, human umbilical vein endothelial cells; Ig, immunoglobulin; IL-l, interleukin-1; LPS, lipopolysaccharide; m, mouse; MRLjl (mouse strain), MRL/Mp-2pr/lpr (MRL strain homozygous for the ‘lymphoproliferative gene’ mutation); nt, nucleotide(s); RT-PCR, reverse-transcription polymerase chain reaction; SP, signal peptide; TM, transmembrane region; TNF, tumor necrosis factor; VCAM- 1, vascular CAM- 1.

1990; Bochner et al., 1991). VCAM-1 appears also to be constitutively expressed in vivo on the stromal cells of the bone marrow (Miyake et al., 199 1) and on cells within the germinal centers of lymph nodes (Freedman et al., 1990). VCAM-1 is a transmembrane protein and a member of the Ig gene superfamily. Initially, VCAM-1 cDNA cloned from activated HUVECs was reported to contain six kinds of Ig units corresponding to six Ig-like domains (Osborn et al., 1989). Subsequently, cDNAs isolated from cytokine-activated endothelium were found to contain an additional Ig unit, and this seventh one was recognized as the major form of human (h) VCAM-1 (Polte et al., 1990; Cybulsky et al., 199 la; Hession et al., 199 1); analysis of hVCAM-1 genomic DNA showed the possibility of additional exons $1 and $2, corresponding to the sequence of rabbit cDNA (Cybulsky et al., 1991b). In order to find sources of VCAM-1 mRNA in the mouse, RNAs were obtained from various tissues after the injection of bacterial LPS (which leads to the systemic release of TNF and IL-l) or from the enlarged lymph nodes of MRL/MP-lpr/lpr (MRL/l) mice, which have been used frequently as an animal model for autoimmune

262 disease (Theofilopoulos and Dixon, 1985; WatanabeFukunaga et al., 1992). A high expression of VCAM-1 mRNA was found in the lymph nodes of MRLjl mice, which were thus used as a source of mRNA to clone mouse (m) VCAM-1 cDNA.

Mouse VCAM-1 cDNA SP

Ig Unit

11213 “i_ .&“”

G_

(2972 bp)

D

A

4 J*

0

3’UTR

5 B

4OObp

-

81-13 Y-11

* $_

EXPERIMENTAL AND DISCUSSION

: cys

: hclone : Primer for PCR

50-12 (A), 6-12 (A)n49-21 (Ah

(a) In vivo expression of VCAM-1 mRNA Fig. 1 is a Northern blot analysis of mouse RNA from various sources using a hVCAM-1 cDNA as a probe. VCAM-I mRNA is expressed in the lung and, to a lesser extent, in the spleen, but only after LPS injection. The highest expression, however, is found in the enlarged lymph nodes of MRLjl mice, where it is constitutive.

Fig. 2. The domain structure of mVCAM-1 cDNA and the position of the 1 clone. Asterisks mark the positions of the Cys residue. Arrows with letters A, B, C, and D show the position of primer for PCR. Sequence of primer A, S-CAACGATCTCTGTACATCCC; primer B, S-AGAGGCTGTACACTCTGCCT; primer C, 5’-AAGGATCCGGTACCAAGCAGAGACTTGAAATGCC; primer D, 5’-CCCTTGAACAGATCAATCTCC. SP, TM, CP, and 3’UTR show signal peptide, transmembrane region, cytoplasmic tail, and 3’-untranslated region.

(b) Screening of cDNA libraries Two kinds of cDNA libraries were produced, by either random hexamer or oligo(dT) priming of poly(A)+RNA from MRLjl mouse lymph nodes and cloning into hgtl 1 phage. From about 1 x lo6 screened plaques, five clones were selected using the DIG System (Boehringer Mannheim) with the hVCAM-1 cDNA probe (Fig. 2).

sequence, the complete protein-coding region of 739 aa, and a 737-bp 3’-untranslated region preceding the poly(A) tail. Three independent clones (50-12, 6-12, and 49-21) showed poly(A) addition at the same position, which corresponds to that of hVCAM-1 (Hession et al., 1991). Clone 6-12, obtained from the random priming cDNA library, had a long poly(A) sequence of over 100 bases. The total nt sequence (2972 bp) of the mVCAM-I cDNA showed a 76.4% similarity with the hVCAM-1 nt sequence. The coding (2220 bp) and 3’-untranslated regions showed 80.4% and 65.1% similarity, respectively. The total aa sequence (739 aa) showed a 75.9% similarity and the same domain structure. The SP, Ig units, TM, and CP showed 70.8%, 74.9%, 81.8%, and 100% similarity with the hVCAM-1 aa, respectively. Despite the 85.2% similarity at the nt level, the two CPs have an entirely identical sequence of 19 aa. This identity has not been observed so far with other CAMS; the CPs of mouse and human intercellular adhesion molecule- 1 (ICAM- l), for instance, show 55.2% and 70.0% similarity at the aa level and nt level, respectively (Siu et al., 1989; Tomassini et al., 1989). Conservation of this sequence suggests that the CP of the VCAM-1 molecule may play an important functional role. The N-terminal sequence of a ‘VCAM-like adhesion molecule’ purified from a murine stromal cell line has been reported (Miyake et al., 1991). This sequence, XXIEISPE, is compatible with that of the mVCAM-I (aa 25-32). Five N-linked glycosylation sites are predicted in mVCAM-1 instead of seven sites in the human one. The cDNAs of three independent clones (8 1- 13, Y- 11, and 50-12) contained seven rather than six Ig units, a result which was further confirmed by RT-PCR of lymph node RNA, showing only the expected 839-bp band (Fig. 4). All Cys residues in the Ig units were conserved

(c)Sequence of

the mouse VCAM-1 cDNA and homology with hVCAM-1 cDNA Fig. 3 shows the complete nt sequence of the mouse VCAM-1 cDNA, consisting of a 5’-flanking 15-bp

1

12345

2

3

-8s

-8s

78s

+lSS

(A)

(B)

Fig. 1. Northern blotting analysis using a hVCAM-1 cDNA probe (Hession et al., 1991). RNAs were subjected to electrophoresis in a 1.1% agarose gel containing 6.6% formaldehyde and then transferred to nylon membranes (Hybond-N, Amersham, UK). Hybridizations were done under stringent conditions with a random-primed “P-labeled hVCAM-1 cDNA. (A) Total RNA (10 pg) obtained from: 1, lymph nodes of a 4-month-old MRLjl mouse; 2, lung and 3, spleen of a BALB/c mouse; 4, lung and 5, spleen of a BALB/c mouse 2 h after LPS intravenous injection (5 ng, E. coli 055:BS DIFCO Lab.). (B) MRLjl mouse lymph node RNA: 1, 10 c(g of total RNA; 2, 1 ug of poly(A)+RNA; 3, 2 pg of poly(A)+RNA. RNAs were isolated from these tissues as described (Chomczynski and Sacchi, 1987).

263 60

is

L F s w TCCAAGTCCG s K s ” ACAGCAACAT T A T c CCCAAGGATC P K D P AAGTGTTTGG K c L A GACCAGCTCR D Q L M AAGRGTTTGG K s L E CGAGCTAAAT R A K L GAACTACAAG E L Q ” CTTCAAGAGG L Q E G ATTTTCTCGG

D I s P GCTGCTRTTG A R I G AATGGGGTGG N G ” ” GAGGATGAAC E D E H RCCCAGGTGG T Q ” E GTGCATGGGA ” H G R CTGGAGATTG L E I E ATGGGCATAR M G I K GATACCGGGA D T G K CCCAAACRGA P K Q R ATCTGGGTCA I w ” s AGTGATGGGA s D G I CAGCCTCTTT Q P L S

R

T

Q

TTCTGACC** L T M GTGGCTCTGG G s G CAGAGATTCA

E

I

Q

CTCCAGACAT P D I TGAACAGACA

N

R

Q

AAGTAACCTT v T F TAC*CATTGA H I D TCTACATCTC Y I s GTGGTGCTGT G A” GCAGGAAGTT

I D s GG*GCCTGTC E P ” GAAGCTGGAA K L E RTTCAGTGGC F s G TTACCCAGTT Y P ” GGAGTTTTCT E F s TACTCCCGTC T P ” CCAAATTGAT Q I D TCCCAGGAAT

P L N A AGTTTTGAGA s F E N CGAAGT*TCC R s I H CCCCTGGAGG P L E ” TACAGGCTGG Y R L P TCAGAAGAGA s E E M ATTGAGGATA I E D I TCTRCACTCA s T L K ACAACG*TCT

s

I p R L- TT TGTTCCAGCG AGGGTCTACC GACAATGACC c s s E T M T G L P AGATAATGAA GTTCTCCAGC TTCTCTCAGG

Q

” A A Q ” G D GCTGTGACTC CCCTTCTTTT TCTTGGAGAA s w R T c D s P s F TAAGGAATGA GGGGGCCAAA TCCACGCTTG s T L ” R N E G A K ACTCTTACCT GTGCGCTGTG ACCTGTCTGC TCLO s Y L c A ” AGGTCTACTC ATTCCCTGAA GATCCAGTG D P ” I F P E v Y s ACAGTCCCTA GACCTGTCAC TGTCAACTCC _T ” P N P v T “S AATTACTGAA GGGGGAGACT ACACTGATGA T L M K L L K G E T AGTCCCTAGA TTGGAAACGA GACCARAATT L E T T s L E T K 1 AATCTCTTGT AGGTTACLCA TTGCCTCGCT s L ” R t H s c L A GGCAGAGTGT ACAGCCTCTT TATGTCAACG Y v N ” Q P L Q S ” GCCCCTCTCC TATACTAGAG GAGGGCAGTC P s P 1 L E E G s P TACC*GCTCC CAAAATCCTG TGGRGCAGAC P A P K 1 L W S R Q CTGAAAATAC TACACTCACC TTCATGTCTA

G

S

ATTTATGTGT I Y v C ATCCAAGTCT I Q" S GGAGACACCG G DT" PAGAAGAAAG K K K R CGCCAGGCAC R Q *Q TCACAATTAA S Q L R TCGCCCGAAC S p EL ATCGTTTRTT s"YF Fp;

GTGARGGGAT E G I CTCCAAAAGR P K D TCATTATCTC I I S CARAGACAGG K T G AGCTGCAGGA LQD GAAGTTTAAC S L T TCCTTGCACT 18 AL TTGCAAGAAA ARK TGTAGCTAAC *

TAACGAGGCT NE A TATACAGCTT 1 Q L CTGCACTTGT c T c AGACRTGGTA D M" TGCCGGCATA RGI ACTTGATGTA L D" CTACTGCGCA YC A AGCTAACATG *NM ATCTGRARTG

s T GGAATTAGCA G I S R ACAGTCTTTC T v F P GGAARTGTGC G NV P TTAAAGTCTG L K S" TACGAGTGTG Y E c E AAAGGRAAAG KG K E TCTTCCTTGG s S LV AAAGGATCGT KGSY TTTGAGAAGA

GATCCTTAAT CTTCCCAGAA

ACTGTTTATT

ACAGCCCCGC

CCCCTCCACA

CCCTTCTCAG

CCAGAAAAGT

TCCCATGATG

TGTATGTAAA TTAGAGTAGC ACTGTTGTAA GCTAACCAGT TTTATTRACR AATGGTAAAG

TAAAGAAGGC TCTGCTTGAC TAACATAAAC

AAAGTGGCTG

AAGGAGCTAA GCGAGGTTGT GCTTGGACTG

ENT

TGACTAGGCA TCCTATGAAT TTTATAGGCA TGGTAGAATG GCTAAGTAAT TCATTGGGGT CTTGTGTAAT TTATAAATAC TATAGTAAAG

TLT

AGCACTCCRT CTTAATGCTT

F

M

AAGTCCCCAT TGTRTATATG ATGCAGGTCA CTTAGGAAAG TATGTTTTAT

GTCACCTTTT ATGATTTCAIT GTTTTTTAAA AGGTCACTGG GTTGACTTTC TTTCTCTACA TGGTACTGTA GACTAACCTG ATTTAAATGC

TGGAAGTTTC

TG

s

” ” CCCAGRCRGA Q T D TGTTGAGCTC L s s AAAGGACRCT RTL TTAAAATGAG K M s ATGTGTATCC ” Y P AGAAATATTT K Y F CCTTCATCCC F I P GTGGTGAAAT G E M TTGCCCCCAA A P K CTGTGAACCT

L T c CAGTCCCCTC s P L TGTGGGTTTT

”

G

F

GGAAAAGAGA EKR

TGGGCCACTT G P L CTTTGACCM F D H TTTGGAGGAA L E E CACCATTGAA T I E GGAAWTGAA E s E GGAAACCACC E T T GACCTGCTCA

LT.cs

“U AGCTA?iATAA L N N CAAAAAGGGA K

Y L c CTACTCTTTC Y s F GGTCACRGTC ” T ” GTTCAAGGGT F K G TCTAGAARCC L E T TCTTGTTTGC L ” c GACTGTCAAA T ” K CTCCACAAGG s T R AGCTCCTGAG A P E A?ATGCCA.CC

II

D

GAAAATCAGT KS" CATCTAAGAG S K S CCGAAACATG ETW TGGATGGCTC D G S RATCTAAGAC S K T AACATAACAA H N K TAATACCTGC I PA ACAGTCTGGT SL" GACATTRTTT AACC?AGCCA

CTTGATCCCT CGTTGAAGTG TAAAATAAAA GAACGTAATC TTTAATATTR TTTCTTCAAC AGAAGTCAGC AGGTACTACT CGGTACGGGG TACTTCATTT

TGGGGAACTG G E L CGRTTCCGGC

s

120 35 180 55 240 75 300 95 360 115 420 135 480 155 540 175 600 195 660 215 720 235 780 255 840 275 900 295 960 315 1020 335 lOS0 355 1140 375 1200 395 1260 415 1320 435 1380 455 1440 d7r.

.._

1500 495

1560 515 1620 535

G

GACTTGTCTT

Fig. 4. RT-PCR analysis of MRLjl mouse lymph node mRNA. (A) Ethidium bromide-stained gel; (B) the same after Southern transfer and hybridization with DIG-labeled mVCAM-1 probe. Samples: 1 and 4, negative control for PCR (template minus); 2 and 5, oligo(dT)-primed cDNA, 3 and 6, random hexamer-primed cDNA. The expected size of PCR product for primers A and B (see Fig. 2) (lanes l-3) is 839 bp (7Ig unit) or 563 bp (6-Ig unit), primers C and D (see Fig. 2) (lanes 4-6) is 493 bp. Methods: PCR products were subjected to electrophoresis in a 1% agarose gel containing 50 mM Trisborate pH 8.3 and 1 mM EDTA and then transferred to nylon membranes (Hybond-N plus, Amersham, UK). Hybridizations were done under stringent conditions with a random-primed DIG-labeled mVCAM-1 cDNA.

1680

555 1740

575 TGAACTGATT 1800 E L I 595 TGTCAAAGAG 1860 v K E 615 G?,TAATCCTG 1920 III, 635 GTACACCATC 1980 Y T I 655 TGAAGTTGGC 2040 E" G 675 GGACTRTTTT 2100 D Y F 695 CATCGGGATG 2160 I GM 715 GGAGGCACAG 2220 EAQ 735 ATAARACCCA 2280 770 TGCATTCAGA 2340 TGCTGAATGC 2400 ACTGGAATGG 2460 TTATGCCGTC 2520 AGAGAGTGTT 2580 ATGTTAATGT 2640 CTAATTTTAT 2700 GTTCTPATATT 2760 TTCTAATTCA 2820 ACCTGTTTCT 2880 D

123456

between the human and mouse forms. Thus, the mVCAM-1 cDNA does not contain the additional domains Ql and Jr2 which were found in rabbit VCAM1 cDNA (Cybulsky et al., 1991b). Fig. 5. shows the aa sequence similarity between each of the Ig units. The first, second, and third units are homologous to the 4th, 5th, and 6th units, as is the case with hVCAM-1. The strong similarity between the 1st and 4th Ig units of hVCAM-1 (76%) is, however, not found with mVCAM-1 (56%). Between m and h VCAM1, the same Ig units (i.e., bearing the same numbers) are highly similar, especially the 7th unit (83%).

2940 2972

Fig. 3. The nt sequence of cDNA encoding the mVCAM-1 and deduced aa sequence. The aa are aligned with the first nt of each codon. The first ATG is at position 16. The asterisk marks the stop codon. The hydrophobic putative signal peptide and transmembrane sequences are underlined in bold. Potential N-linked glycosylation sites are underlined. The EMBL accession No. is X67783. Methods: Five hgt 11 phage clones were isolated from cDNA libraries using the DIG-labeled hVCAM-1 cDNA probe. Hybridization and immunological detection was performed following the manufacturer’s protocol (DIG DNA Labeling and Detection Kit; Boehringer Mannheim). The insert fragments were subcloned in the phagemid vector, pBluescript II SK + or SK- (Stratagene, La Jolla, CA). The nt sequence was determined by the dideoxy chain-termination method using single-stranded phage DNA template and T7 polymerase (Sequenase version 2; US Biochemical, Cleveland, OH).

I

ii 7 1

I

\n \

n 26

I

Fig. 5. The aa sequence similarity between each Ig unit of m and h VCAM-1. Residues of each Ig unit: 1, aa 24-114; 2, aa 115-223; 3, aa 224-309; 4, aa 310-402; 5, aa 403-511; 6, aa 512-598; 7, aa 599-690. Underlined numerals indicate over 50% similarity and n, no similarity,

264 (d) Conclusions (I) The nt and aa sequences of VCAM-I are highly conserved between human and mouse. In particular, the cytoplasmic tail shows an identical sequence of 19 aa, despite having only 85.2% similarity at the nt level; this suggests a critical functional role for it. (2) Three independent h clones had a 7-Ig unit, and not a 6-Ig unit, type cDNA, a finding which was confirmed by RT-PCR. After submission of this paper, we became aware of a report by Hession et al. (1992) describing a similar nt sequence and a 7-Ig unit structure using two clones obtained from mouse lung after LPS injection.

ACKNOWLEDGEMENTS

This work was supported by the Swiss National Research Foundation (31-28866.90) and by a Glaxo IMB fellowship (to K.A.).

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the human VCAM-1 gene. Proc. Natl. Acad. Sci. USA 88 (1991b) 7859-7863. Elites, M.J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M.E. and Lobb, R.R.: VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA4/fibronectin binding site. Cell 60 (1990) 577-584. Freedman, AS., Munro, J.M., Rice, G.E., Bevilacqua, M.P., Morimoto, C., McIntyre, B.W., Rhynhart, K., Pober, J.S. and Madler, L.M.: Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM- 10. Science 249 (1990) 1030-1033. Hession, C., Tizard, R., Vassalo, C., Schiffer, S.G., Golf, D., Moy, P., Chi-Rosso, G., Luhowskyj, S., Lobb, R. and Osborn, L.: Cloning of an alternate form of vascular cell adhesion molecule-l (VCAM-1). J. Biol. Chem. 266 (1991) 6682-6685. Hession, C., Moy, P., Tizard, R., Chisholm, P., Williams, C., Wysk, M., Burkly, L., Miyake, K., Kincade, P. and Lobb, R.: Cloning of murine and rat vascular cell adhesion molecule-l. Biochem. Biophys. Res. Commun. 183 (1992) 163-169. Miyake, K., Medina, K., Ishihara, K., Kimoto, M., Auerbach, R. and Kincade, P.W.: A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. J. Cell Biol. 114 (1991) 557-565. Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., ChiRosso, G. and Lobb, R.: Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59 (1989) 1203-1211. Polte, T., Newman, W. and Gopal, T.V.: Full length vascular cell adhesion molecule 1 (VCAM-1). Nucleic Acids Res. 18 (1990) 5901. Rice, G.E. and Bevilacqua, M.P.: An inducible endothelial cell surface glycoprotein mediates melanoma adhesion. Science 246 (1989) 1303-1306. Rice, G.E., Munro, J.M. and Bevilacqua, M.P.: Inducible cell adhesion molecule 110 (INCAM- 10) is an endothelial receptor for lymphocytes. A CD1 l/CDl8_independent adhesion mechanism. J. Exp. Med. 171 (1990) 1369-1374. Siu, G., Hedrick, SM. and Brian, A.A.: Isolation of the murine intercellular adhesion molecule 1 (ICAM-1) gene. ICAM- enhances antigen-specific T cell activation. J. Immunol. 143(1989) 3813-3820. Theofilopoulos, A.N. and Dixon, F.J.: Murine models of systemic lupus erythematosus. Adv. Immunol. 37 (1985) 269-390. Tomassini, J.E., Graham, D., Dewitt, CM., Lineberger, D.W., Rodkey, J.A. and Colonno, R.J.: cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 86 (1989) 4907-4911. Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A. and Nagata, S.: Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356 (1992) 314-317.