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The EMBO Journal vol.8 no. 5 pp. 1 361 - 1368, 1989
The primary structure of the 4 subunit of VLA-4:homology to other integrins and a possible cell -celladhesion function
Yoshikazu Takada, Mariano J.Elices,Carol Crouse and Martin E.HemlerDana-Farber Cancer Institute, Harvard Medical School, Boston,MA 02115, USA
Communicated by J.-P.Thiery
VLA4 is a cell surface heterodimer in the integrinsuperfamily of adhesion receptors. Anti-VLA-4antibodies inhibited cytolytic T cell activity, withinhibitory activity directed against the effector T cellsrather than their targets. Thus, whereas other VLAreceptors appear to mediate cell-matrix interactions,VLA4 may have a cell-cell adhesion function. Tofacilitate comparative studies of VLA4 and otherintegrins, cDNA clones for the human oe4 subunit ofVLA4 were selected and then sequenced. The 3805 bpsequence encoded for 999 amino acids, with an N-terninus identical to that previously obtained from directsequencing of purified a" protein. The a1 amino acidsequence was 17-24% simnilar to other integrin a chainswith known sequences. Parts of the a(4 sequence mostconserved in other a chains include (i) the positions of19/24 cysteine residues, (ii) three potential divalent cationbinding sites of the general structure DXDXDGXXD and(iii) the transmembrane region. However, ce4 standsapart from all other known integrin a subunit sequencesbecause (i) a'4 has neither an inserted I-domain, nor adisulfide-linked C-terminal fragment, (ii) its sequence isthe most unique and (ill) only ca4 has a potential proteasecleavage site, near the middle of the coding region, whichappears responsible for the characteristic 80 000 and70 000 Mr fragments of aCY.Key words: Integrin/cell adhesion/cytolytic T cell/cDNAsequence
result suggests a possible cell -cell adhesion function forVLA-4.The discovery of the integrin superfamily of cell adhesion
receptors has greatly contributed to our understanding of celladhesion mechanisms (Hynes, 1987). Within the integrinsuperfamily, there are at least 11 distinct receptorssubdivided into three families known as (i) VLA proteins(Hemler et al., 1987b, 1988), (ii) cytoadhesins (Ginsberget al., 1988), and (iii) LFA-1, Mac-I and plS50,95 proteins(Springer et al., 1987). VLA-4 is known to be a memberof the VLA/integrin family because the aC4 subunitassociates with the common VLA f (fll) subunit (Hemleret al., 1987a,b) and the a4 N-terminal amino acid sequenceshowed marked similarity to other integrin a subunitN-terminal sequences (Takada et al., 1987). However,VLA-4 also differs from other integrin heterodimers becausethe VLA-4 a(x subunit is only weakly associated with its j31subunit, and also the 150 000 Mr a'4 protein usuallyundergoes partial 'trypsin-like' cleavage to form 80 000 and70 000 Mr fragments (Hemler et al., 1987a). Among thehuman integrin a subunits, those from a fibronectin receptor(VLA-5) (Argraves et al., 1987; Fitzgerald et al., 1987a),collagen receptor (VLA-2) (Takada and Hemler, 1988),vitronectin receptor (Suzuki et al., 1987), platelet gpIlb/Ella(Fitzgerald et al., 1987a; Poncz et al., 1987), Mac-I(Amaout et al., 1988; Corbi et al., 1988; Pytela, 1988) andp150,95 (Corbi et al., 1987) have been sequenced and shownto have 20-60% conservation of amino acids. These six
IntroductionThe cell surface heterodimer VLA-4 is highly expressed onthymocytes, peripheral blood lymphocytes, monocytes, Tand B cell lines and myelomonocytic cell lines (Sanchez-Madrid et al., 1986; Hemler et al., 1987a,b) but has beenfound in only low amounts on most adherent cells and celllines (Hemler et al., 1987a). Thus VLA-4 is different fromother VLA proteins (VLA-2, VLA-3, VLA-5, VLA-6)which are present in varying amounts on nearly all adherentcells and cell lines. Whereas the latter VLA proteins arereceptors for the extracellular matrix components collagen,laminin and fibronectin (Hynes, 1987; Wayner and Carter,1987; Gehlsen et al., 1988; Sonnenberg et al., 1988), aligand for VLA-4 has not been discovered.
Recently, the mouse equivalent of VLA-4 was implicatedas a lymphocyte homing receptor on Peyer's patch highendothelial venules (HEV) (Holzman et al., 1988). This
a b c d
e f 9 h i
Fig. 1. Sequential immunoprecipitations using anti-VLA-4 antibodies.Extract from "25-labeled HPB-MLT cells was immunodepleted bysuccessive incubations with control antibody, B-5G10 or L25 asindicated at the top of the figure. Then the remaining proteins wereimmunoprecipitated using B-5G10 (lanes a, d and g), B-5E2 (lanes b,e and h) or L25 (lanes c, f and i).
V.Takada et al
Specific 51Cr-Release (cpm)
100 200 300 400 500 600
Fig. 2. Inhibition of cytolytic T cell function by anti-VLA-4antibodies. The mAb L25 (A) and B-5G10 (B) were preincubated withcloned cytolytic T cells for 30 min as indicated and then specific 51Crrelease from target cells was determined in a cytolysis assay asdescribed in Materials and methods. The anti-HLA class I mAbW6/32 was used as a negative control antibody (D) and gave resultssimilar to no antibody (not shown). Also in some experiments L25 (C)or an anti-HLA class II mAb (E) was preincubated with target cellsrather than effector T cells. Each experiment was done at fourdifferent dilutions of antibody ascites fluid, and at each dilutionsamples were analyzed in quadruplicate.
a subunits can be subdivided into two groups which haveseveral distinguishing features (Takada and Hemler, 1988).Most prominently, there are (i) those containing an insertedI-domain and (ii) those which undergo protease cleavage,together with disulfide linkage of the cleaved C-terminalfragment.
In this paper, the role of VLA-4 in cytotoxic T lymphocytefunction is investigated, and a mAb (called L25) which waspreviously known to block cytolytic function (Claybergeret al., 1987), is shown to recognize VLA-4. Also, tofacilitate detailed structural comparisons between VLA-4 andother integrins, the a4 subunit was cloned and sequenced.
Recognition of VLA-4 by the mAb L25The mAb L25 was previously found to block cytolytic Tcell function and also recognized a series of proteins of150 000, 85 000 and 75 000 Mr (Clayberger et al., 1987).Because the sizes of those proteins resembled the VLA-4a!4 subunit and its cleaved fragments, the L25 mAb wasdirectly tested for VLA-4 recognition. As shown (Figure 1),both L25 and the anti-VLA-4 mAbs B-5G10 and B-5E2recognized proteins of similar sizes (lanes a-c). Further-more, when the mAb B-5G10 was used to immunodepleteall VLA-4 (lanes d and e) from an HPB-MLT cell extract,material precipitated by L25 was also removed (lane f).Conversely, preclearing with the mAb L25 (lanes g-i)caused the disappearance of VLA-4 as detected by B-5G10(lane g) or B-5E2 (lane i).
Fig. 3. Immunochemical crossreactivity between a4 and a fusionprotein made from a4 cDNA. Rabbit anti-fusion protein antibodieswere positively selected from crude anti a4 antisera by using fusionprotein-Sepharose as described in Materials and methods. The fusion-protein enriched sera was used to immunoblot the fusion protein itself(lane a) and purified VLA-4 protein (lane b). Also, un-enriched anti-a4 sera was used to immunoblot whole cell lysate from HPB-MLTcells (lane c), fusion protein (lane d), and purified VLA-4 protein(lane e).
Inhibition of cytotoxic T cell function by anti-VLA-4antibodiesTo investigate further the potential role of VLA-4 incytotoxic T cell function, the mAbs L25 and B-5G10 weretested for blocking effects. As shown (Figure 2), fourdifferent concentrations of L25 effectively blocked cytolyticT cell activity down to 30-40% of control levels. Anotheranti-VLA-4 mAb (B-SGlO) was less potent, but also blockedcytolysis (down to 50-60% of control levels). When L25was pre-incubated with target cells rather than with killercells, it had no blocking effect. In another controlexperiment, an anti-HLA class II mAb (LB3. 1) effectivelyblocked killing, as expected, since these cytolytic T cellswere directed against class II targets. Similar blocking resultswere obtained using other CTL clones or uncloned CTL thatrecognize class I targets (data not shown). These resultsconfirm the previous L25 anti-cytolytic blocking results(Clayberger et al., 1987), and suggest that VLA-4 couldhave a cell-cell adhesion role during the association ofcytolytic T cells and their targets.
Identification of cDNA encoding for the a4 subunitTo allow detailed structural comparisons between VLA-4and other integrin a subunits, the a4 subunit was clonedand sequenced. Polyclonal anti-human VLA a4 antiserawas used to select multiple clones from a Xgtl 1 library made
IA. Anti -VLA-4
C. Anti-VLA-4j_ ~ (L25), with targets
g D. Control+ Z HAb (W6/32)
E. Anti-Class IIMAb (LB3.1)
Function and primary structure of VLA-4 ce4 subunit
M P T IS AW GAK C A N PCG EPAAV
N (C) C A P G R P Y N V A T E S A S P5 C H NA
S A A H S H G A N P W AV S A P A N W L A MIA A N P A35' TACAGATS ~ASS'- TAS,A A..A5I,S A A.'SIS '-,SA.'1.A.
A (C) P C K N P G 5 A (C) S 5 G S P N G P (C) C K TA C409 GACP'A.ACG3'7.9'... ASA .ASAAS.'.A ('' .'..A'. S -----A-'--
A N Q W A S P 5 P C N G A V AT (C) GC H P W K N K NA59 SAAAATAAAGC'CI S TF-TC9'SCA, GGAGTSACISS CTGATTTA.GA.ACAGA.AITSASTIAA.G. A.A7AS'IICIS T7.IIA' AS~'-.
H N K P G G (C)YP V P P A P A S K P A A P (C)Y Q563'TTGGAGA.AAA"T'~',A'A7S' AA~~GG.IAA-A'A-SCIGTTTACACAAG-AT--A.TI''AA GG,'A'SSS-CSSIIIA'A
G ENSA S C) QAAG GASP PAYTKADSI A G AP GSS (SISWGIS CTTTTTGTCTACA A7ATAG IA ~AAATAAATACAAGSITTTTT