Gene. 114 (1992) 267-271

© 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

267

GENE 06463

Sequence of the bovine CDl8-encoding cDNA: comparison with the human and murine glycoproteins (Integrins; leukocyte adhesion deficiency; adhesion; deficiency)

Dale E. Shuster, Brad T. Bosworth and Marcus E. Kehrli Jr. U.S. Department of Agriculture - Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit. Ames. 1,4 50010-0070 (USA)

Received by J.L. Slightom: 8 November 1991 Accepted: 2 February 1992 Received at publishers: 25 February 1992

SUMMARY The bovine eDNA (CDI8) encoding CDI8, a ceil-surface glycoprotein involved in multiple leukocyte functions, was sequenced and compared with the human and murine sequences. Portions of the 5'- and 3'-untranslated regions of the nucleotide sequences are conserved among the three species, including a 3' A+T-rich region believed to regulate mRNA stability and translational efficiency. The 2833-bp bovine sequence coded for a protein of 769 amino acids (aa). Overall, the deduced aa sequences were > 80% identical among the three species. The aa 96-389 and those in the cytoplasmic domain were very highly conserved with approx. 95 % aa identity. All Cys residues and potential Asn-glycosylation sites present in the bovine sequence were also present in the human and murine sequences. The aa identity was also found in those regions where mutations were found to cause the genetic disease, leukocyte adhesion deficiency. These data identify functionally important regions of the CDI8 mRNA and protein.

INTRODUCTION The/l 2 integrins, also known as Leu-CAMs, are transmembrane glycoproteins synthesized on the surfaces of leukocytes where they mediate intercellular adhesive interactions (Kishimoto et al., 1989a). These integrins are a group of heterodimers consisting of one of three • subunits, CDI la, CDI lb, or CDI lc, and a fl subunit, CDI8. Many aspects of myeloid and lymphoid cell function, including phagocytosis, endothelial adhesion, and chemotaxis, are

Correspondence to: Dr. M.E. KehrliJr., National Animal Disease Center,

P.O. Box 70, Ames, IA 50010-0070(USA) Tel. (505)239-8462; Fax (515)239-8458. Abbreviations: aa, amino acid(s); bp, base pair(s); CDI8, gene (DNA) encoding CDI8; kb, kilobase(s) or 1000bp; LAD, leukocyte adhesion deficiency; nt, nucleotide(s); ORF, open reading frame; RACE, rapid amplification of eDNA ends; SDS, sodium dodecyl sulfate.

dependent upon the f12 integrins (Arnaout, 1990b). These activities can be regulated by the degree of synthesis of the f12 integrins on leukocyte surfaces. Appropriate stimulation can mobilize intracellular stores contained in the secondary and tertiary granules of myeloid cells resulting in enhanced surface production (Arnaout, 1990b). In addition, the adhesive activity of the f12 integrins can be enhanced by physiological phosphorylation of the intracellular portion of CDI8 (Chatila et al., 1989). The importance of f12 integrins for host defense against infectious agents is exemplified by LAD, a human genetic disease also known as Leu-CAM deficiency. This disease is characterized by a marked reduction in leukocyte f12 integrin production and by multiple deficits in phagocyte function, including an inability to emigrate from the vasculature (Arnaout, 1990a). Patients with this disease are highly susceptible to bacterial pathogens and suffer recurrent infections. The disease results from mutations in the gene encoding CD18 (Kishimoto et al., 1989a; Arnaout

268 -105

GT•TT•C•AGATCcGCGRAGCAC•AGCCTGGTGAAGAGCAGAGCCG•AG••CCTGCcAGTC•AGCTGGG•CACCCCTGCCG•GGTCTCCAGGGCATCC•GGGGA•

ATG CTG CGC Met Leu Arg

3

CAG CGC CCC rAG CTG CTG CTC CTA GCG GGC CTG CTT GCC CTC rAG TCC GTC CTG TCC CAG GAG TGC ACC AAC TAC RAG GTC AGC ACC TGC Gln Arg Pro Gin Leu Leu L e u . L e u A l a GI¥ Leu Leu A l a Leu Gln Ser Val Leu Ser Gln Glu Cvs Thr Asn Tyr Lye Val Ser Thr Cv~

33

CGG GAC TGC ATC GAG TCG GGC CCC GGC TGC GCC TGG TGC CAG AAA CTG ARC TTC ACA GGG CAR GGG GAG CCC GAC TCC ATT CGC TGT GAC Arq Asp Cvg I l e Glu S e r GIF P r o GIF c v s A l a Trp c v s Gln Lys Leu ASh Phe ThE Gly Gln Gly Glu Pro Asp SeE I1e A r g C v ~ A s p

63

ACA CGAGCG GAG CTG CTG TCAARG GGC TGC CCA GCT GAT GAC ATC ATG GAACCC RAG AGC CTC GCT GAG ACC CGG GAC AGC HAG GCG GGC Thr Arq A l a Glu Leu Leu S e t Lys G l y EX& Pro A l a Asp Asp I l e Met Glu Pro Lye Ser Leu A l a Glu T h r Arq Asp Set G l n A l e G l y

93

280

AGT CGG RAG CAG CTG TCC CCA CAG GAA GTG ACG CTC TAC CTG AGA CCA GGT HAG GCA GTT GCG TTC ARC GTG ACC TTC CGG AGG GCCRAG Set Arg Lys Gin Leu Set Pro Gin Glu Val Thr Leu Tyr Leu ~ g Pro GIy Gln A l e V a l A l e Phe Asn V a l T h r Phe A r g /trg A l a Lys

123

370

GGC TAC CCC ATC GAC CTG TAC TAC CTG ATG GAC CTC TCC TAC TCC ATG GTG GAT GAC CTC GTC ARC GTC AAG ARG CTG GGG GGT GAC CTG GIF Tyr Pro lle Asp Leu Tyr Tyr Leu Met Asp Leu SeE Tyr S e r Met Val Asp Asp Leu VaI Asn V a l Lys Lys Leu GIF GIF Asp Leu

153

CTC CGG GCC CTC ~S.T GGC ATC ACC GAG TCG GGC CGC ATT GGT TTC GGG TCC TTC GTG GAC RAG AHG GTG CTC CCC TTC GTC AAC ACG CAC Leu Arg AIa Leu ASh Gly l i e T h r Glu Set Giy Arg I l e GIF Phe GIF S e t Phe VaI Asp Lye Thr V a l Leu P r o Phe V a l Ash T h r H i s

183

550

CCC GAG RAG CTG CCG ~AC CCC TGC CCC ARC AAG GAG ARG GAG TGC CAG CCC CCG TT~ GCC TTC AGG CAC GTG TTG RAG CTC ACT GAC RAC Pro GIu Lye Leu Arg ASh Pro cvs Pro Asn Lye Glu Lye Glu c w Gin Pro Pro Phe Ala Phe Arg Hls Val Leu Lye Leu Thr Asp Asn

213

640

TCC ARA HAG TTC GAG ACA GAA GTC GGG AAG CAG CTG ATC TCG GGG ARC CTG GAC GCC CCT GAG GGT GGG CTG GAC GCC ATG ATG CAG GTG Ser Lye Gin Phe Glu Th~ G1u Val Gly Lys Gin Leu Ile Ser GIF ASh Leu Asp A I a Pro G l u GIF G l y Leu Asp A l e Met Met Gin VaI

243

?30

GCC GCG TGC CCG GAG GAA ATC GGC TGG CGC ART GTC ACC AGG CTG CTG GTG TTC GCC ACG GAC GAT GGG TTC CAC TTT GCG GGC GAT GGA Ala Ala ~ Pro Glu GIu lle GIy Trp Arg ASh Val Thr Arg Leu Leu Val Phe A l e T h r Asp Asp GIF Phe Hie Phe A l a G l y Asp G l y 273

020

ARG CTG GGT GCC ATC CTC ACC CCC ART GAC GGC CGC TGC CAC CTG GRA GAC ARC CTG TAC ARA AGC AGC ARC GAA TTT GAC TAC CCA TCG Lys Leu GIF Ale lle Leu Thr Pro ASh Asp GIy A r g ~ y J L H i s Leu GIu Asp ASh Leu Tyr Lye Ser Set Ash GIu Phe Asp Tyr Pro SeE

303

910

GTG GGC CAG CTG GCA CAC AAA CTG GCA GAA AGC AAC ATC CAG CCC ATC TTT GCA GTA ACC AAG AAG ATG GTG AAA ACG TAC GAG AAG HTG Val Gly Gin Leu Ala His Lys Leu Ala Glu Set Asn lle Gln P r o l i e Phe A l a Val T h r Lye Lye Me~ Val Lye T h r T y r G l u Lye Leu

333

I000

ACA GAG ATC ATC CCC RAG TCT GCA GTC GGG C~G CTG TCT GAA GAT TCC AGG ARC GTG GTG GAG CTT ATH AAG RAT GCC TAC RAT ARA CTG Thr Glu Ile Ile Pro Lye Ser Ala Val GIF Glu Leu SeE GIU Asp S e t Arg Asn Val V a l G l u Leu I l e Lye Ash A l a T y r Asn Lye Leu

363

1090

TCC TCC AGA GTC TTC CTG GAT CAC AGC ACC CTC CCT GAC ACC CTG ARA GTC AHC TAC GAC TCC TTC TGC AGT ARC GGG ~ TCG CAG GTG Set Set ArQ Val Phe Leu Asp His Set Thr Leu Pro Asp Thr Leu Lye Val Thr T y r Asp S e t Phe cv~ S e t Asn G l y Lye S e t Gln VaI

393

1180

GAC HAG CCC AGA GGG GAC TGC GAC GGC GTC HAG ATC AAC GTC CCG ATC ACC TTC HAG GTG AAG GTC ACA GCC ACC GAG TGC ATC CAG HAG Asp Gin Pro Arq GIy Asp F.E~Asp Gly Val Gln Ile Asn Val Pro I l e Thr Phe Gin V a l Lye V e l T h r A l e T h r G l u F , Ea I l e Gin Gln

423

1270

HAG TCC TTC ACC ATC CGG GCG CTG GGC TTC ACG GAC ACG GTG ACC GTG CGG GTC CTC CCC HAG TGC GAG TGC CAA TGC CGG GAC GCC AGC Gln Ser Phe ThE Ile Arg Ala Leu Gly Phe Thr Asp Thr Val Thr Val Arg Val Leu P r o G l n ~,~L~ Glu ~ y ~ Gln e v a Azg Asp A I a S e r

453

1360

AGG GAC GGC AGC ATC TGC GGC GGC AGA GGC TCG ATG GAG TGC GGC GTC TGC AGG TGT GAC GCC GGC TAC ATC GGG ~AG ARC TGC GAG TGC Arg Asp GI¥ SeE lie G~& GIF GI¥ Arg GI¥ Set Met Glu cv~ G l y V a l ~ A r g ~ Asp A l e G l y T y r I l e G l y Lye ASh ~ L L G I u F,y~

483

1450

HAG ACG HAG GGC CGG AGC AGC CAG GAG CTG GAG GGC AGC TGC CGC ARG G&C RAC AGC TCC ATC ATC TGC TCG GGG CTG GGG GAC TGC ATC Gin Thr Gin GIy Arg Set Se~ Gin GIu Leu Giu GIy Set cvs Arg Lye Asp ASh S e r S e r l i e I I e c v s S e t GI¥ Leu G l y Asp c v s l i e

513

1540

TGC GGG HAG TGC GTG TGC CAC ACG AGC GAC GTG CCC ARC ARG ARG ATC TAC GGC HAG TTC TGC GAG TGC GAC ARC GTC RAC TGC ~ CGC c v s Gly Gin c w Val e v a His Thr S e t Asp Val Pro Asn Lye Lye I I e T y r GIF Gin Phe e v e GIu e v a Asp ASh V a l Asn cvA Glu Arg

543

1630

TAC GAH GGC CAR GTC TGC GGG GGC GAA RAG AGG GGG CTC TGC TTC TGC GGC ACC TGC AGG TGC GAC GAG HAG TAT GAG GGT TCG GHA TGC T y r Asp Gly Gin Val ~ y £ G l y GIy Glu Lye Arg GIy Leu E~a Phe eva Gly T h r G ~ £ A r g ~kyJIASp Glu Gin T y r G l u G l y SeE A I a ~jy£

573

1720

CAG TGC CTC RAG TCC ACT HAG GGC TGC CTC AAC TTG GAC GGC GTC GAG TGC AGC GGC CGC GGC CGA TGC CGC TGC ART GTG TGC C&G TGC Gin ~y£ Leu Lye Set Thr Gin G l y ~ y J I L e u Ash Leu Asp G l y V a l G l u ~ y £ S e r G l y A r g GIF k r g ~ k r g cv~ ken V a l c ~ . G l n ~ y £

603

1010

GAC CCC GGC TAC HAG CCG CCC CTG TGC AGC GAG TGC CCG GGC TGC CCC GTG CCC TGT GCG GGC TTC GCC CCC TGC AHA GAG TGC CTG RAG Asp Pro G l y Tyr Gin ~ r o P r o L e u G . y ~ S e t G I u ~ £ ~ r o GIF E~£ P r o VaI P r o F,E a k l a GIF Phe A l a P r o ~ E a T h r G l u c ~ - Leu Lye

633

1900

TTC GAC ARG GGC CCC TTC GCC AAG ARC TGC AGC GCA GCG TGC GGG HAG ACG ARG CTG CTG TCC AGC CCG GTG CCC GGC CGC RAG TGC RAG Phe Asp Lys Gly Pro Phe AIa Lys Asn~;:~& Ser AIa M a GIF Gin Thr Lye Leu Leu S e t S e t P r o V a l P r o G l y Azg Lye E~£ Lye

663

1990

GAG CGC GAC TCC GAG GGC TGC TGG ATG ACC TAC ACC CTG GTG CAG CGC G&C GGG CGG GAC AGA TAC GAC GTG CAC GTG GAC GAC ATG CTC Glu Arg Asp Set GIu Gly cv~ Trp Met ThE Tyr ThE Leu VaI Gin Ar9 Asp G l y A t 9 Asp Arg T y r Asp V a l H i s V a l Asp Asp Met Leu

693

2080

GAG TGT GTG RAG GGC CCC ARC ATC GCT GCC ATC GTG GGG GGC ACC GTG GGG GGC GTC GTG CTC GTC GGC ATC CTC CTG CTG GTC ATC TGG Glu Cv~ VaI Lye Gly Pro Ash lie Ale Ale !le Val Gly Gly Thr Val GIy Gly Val Val Leu Val Gly lie Leu Leu Leu Val lle Trp

723

2170

RAG GCC CTG ACA CAC CTG AGC GAC CTH AGG GAG TAC CAT CGC TTT GAG RAG GAG ARG CTC ARG TCC HAG TGG ARC ARC GAT ARC CCT CTT Lys A l e Leu Thr Hie Leu S e t Asp Leu Rrg Glu Tyr H i s Arg Phe GIu Lye Glu Lye Leu Lye Ser Gin Trp ASh ASh Asp ASh Pro Leu

753

2260

TTC RAG AGT GCC ACC ACG ACA GTC ATG ARC CCT RAG TTT GCC GAG AGT TAG GGGTGCCCGGTGAAGACAAGGCCTTCTGCACCACCCAGATGGGRACACCCC Phe Lye SeE A i a Thr Thr Thr VaI Met Asn Pro Lys Phe A l e Glu Set * 769

I0

I00

19o

460

2363

CTCTC~&CGTCCCCTCCAGCAGGCTGACCGTG&CCCCGCTGcCTCGTGGACGTGGCTG&CAACTTCACCGTT/~%CC~ATGC&CTGCTTTTTCTGCCCCAG~ATGATGGGCGTGGCc

2481

AGGTTATTCT&TGGGCTCATGGT/~GGGCCAGCCTACCCCTTCTGATATGAATGACTTTTGATAGC/~AGTCAGA~t%GG~ATTGCCTACATTTTGTATGGTTACAC&C&GGTCCTTTGT

2600

AA~/~TTAGTACAGr-AGTCTG~TGAAGA~TTATTTATGTGTGA~CTTCTCAGGGTATGA~GTTACATCCCCTTGGTTATGCTGCCCCCA~TCAATAAAA~AAA~AATCRAT~AN~ AJUUtqJUU~ 2728

2719

Fig. I. T h e nt a n d d e d u c e d a a s e q u e n c e s for b o v i n e CDI8 c D N A . T h e 55 C y s r e s i d u e s are u n d e r l i n e d . T h e p u t a t i v e l e a d e r p e p t i d e ( y o n Heijne, 1986) a n d t r a n s m e m b r a n e regions are d o u b l e u n d e r l i n e d . T h r e e p h a g e ). c l o n e s w e r e s u b c l o n e d a n d c o m p l e t e l y s e q u e n c e d in b o t h d i r e c t i o n s ( S a n g e r et al., 1977). T h e s e c l o n e s w e r e partial length a n d c o n t a i n e d the p o l y ( A ) tail. T h e 5' e n d w a s amplified using t h e R A C E p r o c e d u r e ( F r o h m a n et al., 1988). P o l y ( A ) ÷ R N A

269 et al., 1990). Recently, LAD disease has been identified in cattle (Kehrli et al., 1990). However, the//2 integrins had not been well characterized in ruminants, and the gene for CD 18 needed to be sequenced before mutations in this gene could be identified.

EXPERIMENTAL AND DISCUSSION

kb

-

3.0

(a) CDI8 cDNA cloning and sequencing Six independent, hybridizing clones were isolateo when 150000 Agtll plaques from a bovine concanavalin A/phorbol myristate acetate-stimulated lymphocyte eDNA library (ATCC, RockviUe, MD) were screened with a fulllength murine CDI8 eDNA probe (Wilson et al., 1989). One clone was identified from 300 000 ~.gt10 plaques from a bovine spleen eDNA library (Clontech Laboratories, Palo Alto, CA). The longest clone, 2.7 kb, lacked the 5'untranslated region and the first 60 bp of the coding region, based upon comparison with the human and murine sequences. The 5' end of the gene was amplified using the RACE procedure (Frohman et al., 1988). The eDNA sequence for bovine CDI8 contains 2833 bp with an ORF of 2310 bp that codes for 769 aa followed by 418 bp in the 3'-untranslated region (Fig. 1). Compared to human CDI8 (Kishimoto et al., 1987), bovine CDI8 contains a 22-aa putative leader peptide, a Cys-rich region between aa 444 and 633, a hydrophobic transmembrane region, and a cytoplasmic tail. The region immediately preceding the ATG start codon is similar in the bovine, human, and mouse. This region contains the sequence A G ( n ) A C where n -- 5 in the mouse (Wilson et al., 1989), n = 4 in the bovine, and n = 3 in the human (Kishimoto et al., 1987). The 8-mer 5'-TTATTTAT sequence, which is found in the 3'-untranslated region of murine CDI8 cDNA (Wilson et ai., 1989) and in a similar location of mRNAs that encode many other inflammatory proteins (Caput et al., 1986), is also present in the 3'-untranslated region of the bovine gene (nt 2629-2636). The 7-mer 5'-TTATTTA sequence is present in the 3'-untranslated region of human CD18 (Kishimoto etal., 1987). This 3'-untranslated sequence may regulate mRNA stability and translational efficiency to coordinate enhanced translation in response to inflammatory stimuli (Shaw and Kamen, 1986; Kruys et al., 1999). Northern-blot analysis of total RNA from unst'mulated peripheral blood leukocytes revealed a CD18 transcript size of approx. 3.0 kb (Fig. 2), a length similar to that of the

Fig. 2. Northern-blot analysis of bovine CDI8. Total RNA (10 Fg) was electrophoresed in a I °h agarose gel containing formaldehyde, transferred to a nylon membrane, and then hybridized with the 2.7-kb bovine CDl8 eDNA clone labelled with [a.~2P]ATP (Fcinberg and Vogelstein, 1983) in 50% formamide/5 x Denhardt's/5 x SSPE/0.5% SDS/0. I mg per ml of salmon sperm DNA at 42 °C overnight. The membrane was washed twice in 6x SSPE/0.5% SDS at 25°C and twice in 1 x SSPE/0.5% SDS at 37°C. SSPE is 150 mM NaCI/10 mM NaH2PO4/I mM EDTA pH 7.4. Transcript size (3 kb; see righthand margin) was compared with 18S and 28S ribosomal bands.

human transcript (Kishimoto et al., 1987) and comparable in size to the 2833-bp eDNA sequence.

(b) Deduced aa sequence comparison among species The deduced aa sequence of bovine CDI8 contained exactly the same number of aa as human CD 18 and one fewer than murine CDI8 (Fig. 3). The bovine sequence shares 83 and 82% identity with the human and murine sequences, respectively. Conservation of aa is very high in the cytoplasmic domain and in the extracellular region from aa 96-389, where aa identity is approx. 95 %. This extracellular portion includes a region of approx. 245 aa that is similar among several integrin/l subunits (Kishimoto et al., 1989a; Arnaout, 1990b). Four conserved Cys-rich domains are present in the extracellular portion of human CDI8 at aa 449-628 (Kishimoto et al., 1987). These same domains are present in the bovine and murine proteins. Every Cys residue in the bovine sequence is present in the other species. One Cys of human CD18, present in the leader peptide at aa 19, is absent from bovine and murine CDIS. Of six potential Asn-glycosylation sites in human CD18, the one at aa 212 is not present in the bovine or murine sequences. The high aa identity among species corroborates

from leukocytes was reverse transcribed with a CDi8-specific primer (antisense to nt 712-734), purified, and tailed with dATP. The tailed cDNA was then amplified using a nested polymerase chain reaction. The first amplification utilized 0.6 #M primer (5'-GACTCGAGTCGACAAGCTTTTTTTTTTTTVfTTT) and 0.3 FM CD18 specific primer (anti-sense to nt 563-586), and the second amplification utilized 0.6 FM dT primer and 0.3 FM internal CDI8 specific primer (anti-sense to nt 237-259). RACE products were cloned and sequenced. Sequence data have been deposited with GenBank under accession No. M81233.

270 MLRQRPQLLLLAGLLALQSVLSQECTNYKVSTCRDCIESGPGCAWCQ~TGQGEPDSIRCDTRAELLS --GL--P--A-V---S-GC ....... KF---S--E ........ T ......... P-D PQ--M --GPHSL--A .... FF-G-AV ..... K .... S ..... Q ..... S P ..... L ...... Q--L

140

KGCPADDIMEPKSLAETRDSQAGSRKQLSPQEVTLYLRPGQAVAFNVTFRRAKGYPIDLYYLMDLSYSMV R--A ..... D-T ..... QEDHN-GQ ...... ......... D-R-I-NPEFD-R-Q .......

KK-

A A

........... .............

L L

210

DDLVNVKKLGGDLLRALNGITESGRIGFGSFVDKTVLPFVNTHPEKLRNPCPNKEKECQPPFAFRHVLKL ---R---N

E

Bovine Human Murine

D

Q---E

A 280

TDNSKQFETEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWBNVTRLLVFATDDGFHFAGDGKLGAILT -N--N--Q ....

N--O

.

.

.

.

.

.

.

.

.

.

I-

PNDGRCHLEDNLYKSSNEFDYPSVGQLAHKLAESNIQPIFAVTKKMVKTYEKLTEIIPKSAVGELSEDSR R N SR, S M--R. S D--S

350

NVVELIKNAYNKLSSRVFLDHSTLPDTLKVTYDSFCSNGKSQVDQPRGDCDGVQINVPITFOVKVTATEC ---H NA ................ VTHRN __-Q ...... y . . . . . . A-SIGKS N-V ......

420

M-S-490

IOO0SFTIRALGFTDTVTVRVLPOCECQCRDASRDGSICGGRGSMECGVCRCDAGYIGKNCECQTQGRSS --E---V ........ I---Q ....... ,---,---'-'-'-'-''---' .... T----E---V--Q-R. Q--EQ-L---K-V .... I---ES QELEGSCRKDNSSIICSGLGDCICGQCVCHTSDVPNKKIYGQFCECDNVNCERYDGQVCGGEKRGLCFCG N .......... V .... L ....... G-L .... Y .... TI ..... N ...... PG ....... .... RN ........ V--E-F--Y ........... NS ..... SD--S-N--

560

TCRCDEQYEGSACQCLKSTQGCLNLDGVECSGRGRCRCNVCQCDPGYQPPLCSECPGCPVPCAGFA K---HPGF ....... ERT-E .... PRR E-HS---L---Q ...... S--GKYI K-S-KPG QR--T .... ARL ....... H-Q--R-I--E ..... M-ED--S-GSH-RDNHTS-A

629

PCT S-A

ECLKFDKGPFA~NCSAACGQTKLLS SPVPGRKCKERDSEGCWMTYTLVQRDGRDRYDVHVDDMLECVKGP ..... E .... G ....... PGLQ-SNN--K--T .......... VA---E-Q--M---LIY--ESR---A-E .... VQ-AGMT-QTI-LKKKP .......... I .... Q-K---NI-NI--E-S .......

699

NIAATVGGTVGGVVLVGILLLVIWKALTHLSDLREYHRFEKEKLKSQWNNDNPLFKSATTTVMNPKFAES A-I--X ........... I ........ R -V ........ V .... I-V ............ T ..... R

769

Fig. 3. Comparison of deduced aa sequences (single letter designation) Ibr bovine (Fig. [ ), human (Kishimoto et al., 1987), and murine (Wilson et al., 1989) CDI8. A dash denotes aa identity with the bovine sequence. A space was inserted after aa 626 in bovine and human sequences to maintain maximal identity. Potential Asn(N)-glycosylation sites arc in bold.

earlier observations that monoclonal antibodies are crossreactive (Kehrli et al., 1990). Kishimoto et al. (1989b) identified one cause of LAD as a 90-bp deletion in CDI8 mRNA resulting from aberrant splicing. The 30 aa (332-361) coded by this region are highly conserved among the three species. Arnaout et al. (1990) discovered two different point mutations in the human CDI8 gene which cause LAD. These mutations caused a Lys--Thr substitution at aa 196 and an Arg~Cys substitution at aa 593. The Lys and Arg at these positions are conserved in all three species. The CD 18 glycoprotein must interact with the ~ subunits to form the//2 integrins. The/~., integrins then bind to endothelial adhesion molecules to mediate leukocyte-endothelial adhesion (Kishimoto et al., 1989a). This adhesion allows neutrophils to migrate into areas of infection or inflammation. The high degree of conservation between aa 96-389 indicates that this region may be important for these molecular interactions. The high degree of conserva-

tion in the intracytoplasmic portion of CDI8 with many Ser, Thr, and Tyr residues may be consequent to the important role that phosphorylation of these residues plays in regulating adhesive activity (Chatila et al., 1989).

ACKNOWLEDG EM ENTS

The authors thank Dr. Ray Wilson for the murine CDI8 cDNA clone.

REFERENCES

Arnaout, M.A.: Leukocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol. Rev. 114 (1990a) 145-180. Arnaout, M.A.: Structure and function of the leukocyte adhesion molecules CDI I/CDI8. Blood 75 (1990b) 1037-1050. Arnaout, M.A., Dana, N., Gupta, S.K., Tenen, D.G. and Fathallah,

271 D.M.: Point mutations impairing cell surface expression of the common fl subunit (CD 18) in a patient with leukocyte adhesion molecule (Leu-CAM) deficiency. J. Clin. Invest. 85 (1990)977-981. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S. and Cerami, A.: Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Prec. Natl. Acad. Sci. USA 83 (1986) 1670-1674. Chatila, T.A., Geha, R.S. and Arnaout, M.A.: Constitutive and stimulusinduced phosphorylation of CD I 1/CD 18 leukocyte adhesion molecults. J. Ce!! Biol. 109 (1989) 3435-3444. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132 (1983) 6-13. Frohman, M.A., Dush, M.K. and Martin, G.R.: Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Prec. Natl. Acad. Sci. USA 85 (1988) 8998-9002. Kehrli Jr., M.E., Schmalstieg, F.C., Anderson, D.C., Van der Maaten, M.J., Hughes, B.J., Ackermann, M.R., Wilhelmsen, C.L., Brown, G.B., Stevens, M.G. and Whetstone, C.A.: Molecular definition of the bovine granulocytopathy syndrome: identification of deficiency of the Mac-I (CDllb/CDI8)glycoprotein. Am. J. Vet. Res. 51 (1990) 1826-1836. Kishimoto, T.K., O'Connor, K., Lee, A., Roberts, T.M. and Springer,

T.A.: Cloning of the fl subunit of the leukocyte adhesion proteins: homology to an extracellular matrix receptor defines a novel supergene family. Cell 48 (1987) 681-690. Kishimoto, T.K., Larson, R.S., Corbi, A.L., Dustin, M.L., Staunton, D.E. and Springer, T.A.: The leukocyte integrins. Adv. Immunol. 46 (1989a) 149-182. Kishimoto, T.K., O'Connor, K. and Springer, T.A.: Leukocyte adhesion deficiency: aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype. J. Biol. Chem. 264 (1989b) 35883595. Kruys, V., Marinx, O., Sham, G., Deschamps, J. and Huez, G.: Translational blockade imposed by cytokine-derived UA-rich sequences. Science 245 (1989) 852-855. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Prec. Natl. Acad. Sci. USA 74 (1977) 546354~7. Shaw, G. and Kamen, R.: A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46 (1986) 659-6,57. yon Heijnc, G.: A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14 (1986)4683-4690. Wilson, R.W., O'Bricn, W.E. and Beaudet, A.L.: Nucleotide sequence of the cDNA from the mouse leukocyte adhesion protein CD 18. Nucleic Acids Rcs. 17 (1989) 5397.