Isolation and chemical characterization of the human B29 and mb-1 proteins of the B cell antigen receptor complex

iMolecuiar ~r)~~~~?o/og.~,, Vol.31,No.6,pp. 419---4X, 1994 Pergamon

Elsevier Science Ltd Printed in Great Britain 0 16 i -5890;94 $7.00 + 0.00

~161-5890(94)E~22-R

ISOLATION AND HUMAN B29 AND

STEFANVASILE,~~

CHEMICAL CHARACTERIZATION mb-1 PROTEINS OF THE B CELL RECEPTOR COMPLEX*

JOHN E.COLIGAN,§ MINORUYOSHIDAT

OF THE ANTIGEN

and BEN K. SEON~//

tDepartment of Molecular Immunology, Roswell Park Cancer Institute,7 Buffalo, NY 14263, U.S.A.; and SLaboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A. (First received 7 June 1993; accepted in revised form 24 January

1994)

Abstract-A

disulfide-linked heterodimeric antigen from human B leukemia cells was detected by radioimmunoprecipitation and Western blot analysis using a monoclonal antibody (mAb). The mAb was generated against a cell membrane antigen preparation from human B prolymphocytic leukemia cells and found to define an extracellular epitope of the smaller component (fl chain) of a heterodimeric antigen on human B leukemia cells. The antigen from BALL-l, a human B leukemia cell line, and fresh (uncultured) B prolymphocytic leukemia cells was found to consist of a 4449 kDa (01chain) and a 36-40 kDa (p chain) component. An additional minor component of 34 kDa was detected in the reduced antigen from BALL-l. For chemical identification of the antigen, we isolated the antigen from a Triton X-100 lysate of BALL-l by immunoaffinity chromatography using the mAh. Determination of the amino-te~inal amino acid sequences of the ft and @ chains unequivocally identified them as the human mb-1 and B29 proteins, respectively. The sequence analyses indicate the molecular heterogeneity of the mb-1 protein and also perhaps the heterogeneity of the B29 protein. We detected three forms of the mb-1 protein which share an identical amino-terminal amino acid sequence and probably two forms of the B29 protein which share an identical amino-terminal sequence. Our sequence data allowed us to establish the authentic amino-terminal amino acid sequence of the human B29 protein which is different from those proposed by others based on cDNA sequence analyses. Comparison of the amino-terminal sequences of the human mb-1 and B29 proteins with those of the mouse mb-1 and B29 proteins showed that the majority of the conserved amino acids in the mb-1 proteins are hydrophobic amino acids. Such conservation of hydrophobic amino acids is not observed in the amino termini of the human and mouse B29 proteins. A ~-tubulin-like protein was found to be co-purified with the mb-l-B29 antigen in the present study. In addition, we found that there is a strong amino acid sequence homology between the microtubule-binding domains of certain human microtubule-associated proteins and an intracellular segment of the human mb-1 protein. Keq’ words: B29 protein,

mb-1 protein,

B cells, antigen

*This work was supported by American Cancer Society grant CH-514. SPresent address: Division of Biomedical Marine Research, Harbor Branch Oceanographic Institution, 5600 U.S. 1 North, Fort Pierce, FL 34946, U.S.A. IlAuthor to whom correspondence should be addressed at: Department of Molecular Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets. Buffalo, NY 14263, U.S.A. fiA unit of the New York State Department of Health. Abbreviations: mIg, cell membrane immunoglobulin; PLL, prolymphocytic leukemia; ALL, acute lymphoblastic leukemia; AEBSF, 4-(2-aminoethyl)-benzenesuIfonylfluoride; CAPS, 3-(cyclohexylamino)I-propanesulfoni~ acid; PVDF, polyvinylidene difluo~de; DTT, dithiothreitol; MT, microtubule; MAP, microtubule-associated protein.

receptor

complex,

/?-tubulin.

Binding of antigen to the antigen receptor on B cells initiates complicated signal transduction cascades which may lead to proliferation, differentiation or programmed cell death of the B cells. Furthermore, cross-linking of the antigen receptor by anti-Ig antibodies results in rapid tyrosine phosphorylation of several proteins (Campbell and Sefton, 1990; Gold ef al., 1990; Clark and Lane, 1991; Takagi et al., 1991; Cambier and Campbell, 1992; Reth, 1992). It has been known for over 20 years that mIg constitutes antigen receptors on B cells (Pernis et al., 1972). The isotype of mIg is IgM or IgM plus IgD on most B cells, but IgG, IgA or TgE can also be found on some B

419

S. VAsn.E et

420

cells. Recently, two disulfide-linked transmembrane molecules, Ig-a and Ig-p, have been defined as integral components of the antigen receptors on murine B cells [reviewed in Cambier and Campbell (1992) and Reth (1992)], The mIg associated @ heterodimer is believed to be important for signal transduction. The murine Ig-x and Ig-b were identified as the products of the mb-1 and the B29 genes, respectively (Hermanson et al., 1988; Sakaguchi et al., 1988; Hombach et al., 1990a, h). Human homologues of murine mb-1 and B29 proteins were detected by immunoprecipitation of the human proteins (van Noesel ef al., 1991; Mason et al., 1991; Nakamura et al., 1992). Recently, in addition, nucleotide sequences of cDNAs encoding human homologues of the murine mb-1 and B29 proteins were reported (Ha et nl., 1992; Miiller et al., 1992; Yu and Chang, 1992; Hashimoto et al., 1993). However, chemical properties of the protein products of the human mb-I and B29 genes have not been well characterized. In this study, we have carried out structural analyses of a disulfide-linked heterodimeric antigen which was defined by our recently described mAb termed SN8 that reacts with an extracellular epitope of this antigen (Okazaki et al., 1993). The results clearly showed that the two components of the antigen are the human homologues of the murine mb-1 and B29 proteins and the epitope defined by SN8 is on the B29 chain of the complex. In addition, amino acid sequence analyses of the human mb-1 and B29 proteins clearly establish the demarcation line between the leader and extracellular domain sequences in the mb-1 and B29 proteins that were deduced by others on the basis of cDNA sequences. In the present study, a ~-tubulin-like protein was found to be co-purified with the human mb-l-B29 complex. The potential significance of this finding is discussed in the text. MATERIALS

al.

Peripheral blood cells derived from a patient with B PLL were obtained at the Roswell Park Cancer Institute clinics as described previously (Takeuchi et al., 1991). This B PLL cell specimen expressed mIgM-K (Okazaki et al., 1993). AminoLink coupling gel, a 4% cross-linked beaded agarose support, was purchased from Pierce (Rockford, IL). Enhanced chemiluminescence (ECL) Western blotting detection reagents and peroxidaselabeled goat anti-mouse Ig were obtained from Amersham (Arlington Heights, IL) and BoehringerMannheim (Indianapolis, IN), respectively. Pansorbin and AEBSF, a new inhibitor of serine proteases, were purchased from Calbiochem (La Jolla, CA). Trasylol and leupeptin were obtained from Sigma (St Louis, MO).

FACS analysis mIgs on BALL-l were determined by FACS analyses using a Becton Dickinson FACScan and murine mAbs specific for human Ig K, A or p as well as FITC conjugated goat F(ab’), anti-human Ig 6 or a. The analyses with murine mAbs and murine control IgG were carried out by an indirect method as described previously (Yokota et al., 1993) using FITC-labeled sheep F(ab’), anti-mouse IgG as the secondary antibody reagent. Analyses with FITC conjugated goat anti-human Ig reagents were carried out as described below. BALL-I was preincubated for 30min at 4°C with RPMI 1640 medium containing 25 mM HEPES, 10% normal goat serum (heat-inactivated), 0.5% BSA, Trasylol (50 KIU/ml) and 0.1% NaN, to reduce nonspecific binding of antibody reagents. The prein~ubated cells were allowed to react with FITC conjugated goat F(ab’)? anti-human Ig for 1 hr at 4°C. Then, the cells were washed three times with PBS containing 0.1% BSA and 0.1% NaN, and subjected to FACS analysis.

AND METHODS

Anrihodies, cells and reagents mAb SN8 was recently generated in our laboratory by immunizing mice with a glycoprotein antigen preparation isolated from the cell membranes of B PLL cells (Okazaki et al., 1993). SN7 (Okazaki, Haruta and Seon, manuscript in preparation), SNll (Takeuchi and Seon, manuscript in preparation), B3-3D1 (anti-human MHC I mAb) (Takeuchi et al., 1991) and an isotype-matched (IgGl-rc) control murine IgG (MOPC 195 variant) (ibid) were also generated in our laboratory. Murine mAbs specific for human Tg ICor ;1 light chain or p heavy chain were purchased from AMAC, Inc. (Westbrook, ME). FITC-conjugated goat F(ab’)? anti-human Ig 6 or cx heavy chain from CALTAG Labs (South San Francisco, CA) were kindly provided to us by Dr Carleton Stewart of our Institute. FITC conjugated sheep F(ab’)? antimouse IgG was purchased from Sigma (St Louis, MO). BALL-l, a B ALL cell line (Miyoshi et al., 1977), was cultured in RPMI 1640 medium supplemented with 4% FCS, penicillin (100 units/ml) and streptomycin (50pgg/ml) as described previously (Seon et al., 1983).

1.65 x IO”’ BALL-l cells were lysed for 1 hr at 4’C, with gentle shaking, in 160 ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl, 1% (vol/vol) Triton x-100, 1mM iodoacetamide, Trasylol (100 KIU/ml), I p M leupeptin and 0.1 mM AEBSF. The lysate was centrifuged at 3OOOg for 20min at 4°C. Sodium deoxycholate was added to the supernatant to a final concentration of 0.5% and the supernate was centrifuged at 150,0001: for 60 min at 4-C. The resulting supernate was applied to three serially connected immunoadsorbent columns. These immunoadsorbents consisted of MOPC 195 variant (4 ml gel; 2.1 mg IgG/ml gel), anti-HLA class I mAb (B3-3Dl) (5 ml gel; 3.7 mg IgG/ml gel), and mAb SN7 (6 ml gel; 3.3 mg IgG/ml gel), all coupled to AminoLink coupling beaded agarose gel (Pierce). The pass-through materials from the three columns were applied to two serially connected immunoadsorbent columns consisting of MOPC 195 variant-agarose (4 ml gel; 2.1 mg IgG/ml gel) and mAb SNX-agarose (4 ml gel; 1.5 mg IgG/ml gel). The columns were washed with 20 mM Tris-HCl, pH 8.0. containing

Human

B29 and mb-1 proteins

0.15 M NaCl and 1% Renex 30, a nonionic detergent. Then, the columns were disconnected and the materials bound to individual columns were separately eluted with 0.1 M glycine-HCl buffer, pH 3.0, containing 0.5% Renex 30 with the eluate immediately neutralized by collecting 2 ml fractions into tubes containing 120 ~1 of 1 M Tris-HCl, pH 8.0. Renex 30 does not have significant optical absorbance at 280 nm and therefore allows fractionated proteins to be detected spectrophotometritally. The materials eluted from the SN8 column were subjected to the third round of immunoaffinity chromatography using only the SN8 column. Radioimmunoprecipitation

and SDS-PAGE

Protein samples were radiolabeled with lz51 by using IODO-GEN (Pierce)-coated Minisorp tubes as described previously (Haruta and Seon, 1986). Immunoprecipitation of ‘251-labeled UP heterodimer from the radiolabeled samples was carried out using SN8 conjugated to AminoLink agarose or Pansorbin which was coated with rabbit anti-mouse IgG and SN8. An isotype-matched (IgGl-lc) control IgG (MOPC 195 variant) or an isotype-matched irrelevant mAb (SNll) that was coupled to an appropriate matrix (AminoLink agarose or Pansorbin) was used as controls. Details of the procedure for immunoprecipitating radiolabeled antigens using Pansorbin coated with rabbit anti-mouse IgG and an IgGl mAb were previously described (Haruta and Seon, 1986). The procedure for immunoprecipitating antigens using SN8-agarose is briefly described below. An aliquot of a radiolabeled sample was pretreated with an irrelevant mAb-agarose by incubating the reaction tube for 2.5 hr in ice-water. The incubated mixture was centrifuged and the supernate was divided into four equal aliquots. Reaction tubes containing these aliquots were incubated with SN8-agarose (in duplicate) or MOPC-agarose (in duplicate) for 4 hr in ice-water and centrifuged. The resulting pellets were washed twice with Tris-HCl, pH 8.0, containing Trasylol (100 KIU/ml), 2 mM EDTA, 0.05% NaN,, 0.1% BSA and 0.5% taurocholate (Tris-BSA-TC) and twice with TrissBSARenex (Tris-BSA containing 0.5% Renex 30 instead of 0.5% taurocholate). The pellets were finally washed with 62.5 mM TrisHCl buffer, pH 6.8. The radiolabeled antigens present in the washed immunoprecipitates were released from the agarose beads by boiling for 3 min in the presence of 2% SDS with or without 0.2 M DTT. The released antigens were analysed by SDS-PAGE as described previously (Haruta and Seon, 1986). Western

blot anlysis

A recently reported procedure (Okazaki et al., 1993) was modified as described below. A sample in 20 mM TrissHCl, pH 7.7, containing 0.5% taurocholate is mixed with an equal volume of sample treatment buffer and incubated for 1 hr at 37°C; the sample treatment buffer consists of 62.5 mM Tris-HCl, pH 6.8,O. 1% SDS, 10% glycerol and 0.006% bromophenol blue. For reduction of the sample, 0.2 M DTT is included in the

421

buffer. The SDS-PAGE separated proteins are electroblotted in 10 mM CAPS, pH 11.0, containing 10% methanol to a PVDF membrane (Immobilon-P, Millipore), for 45 min at 500 mA. The membrane is incubated overnight at 4°C in 0.01 M PBS, pH 7.0, containing 1% nonfat dry milk (blocking buffer). From this point, all steps are carried out at room temperature. The blocked membrane is incubated for 1 hr with SN8 ascites that was diluted lOOO-fold with blocking buffer containing 0.1% Tween 20. The membrane is next washed three times (10 min each) with blocking buffer containing 0.5% Tween 20. Secondary antibody (horseradish peroxidase conjugated goat anti-mouse Ig), which was diluted lOOO-fold with blocking buffer containing 0.1% Tween 20, is incubated with the membrane for 1 hr. Finally the membrane is washed six times for 10 min each in 50 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 0.1% NP-40 and 0.05% Tween 20. Proteins recognized by SN8 are visualized on Kodak X-OMAT AR film using enhanced chemiluminescence (Amersham) following the manufacturer’s protocol. Amino

acid sequence determination

The eluate from the SN8 immunoadsorbent column (see above) was placed in Spectrapor dialysis tubing (MWCO 50,000) (Spectrum, Los Angeles, CA) and dialysed for a total of 6 days against four daily changes of 20 mM Tris-HCl buffer, pH 8.0, containing 0.5% deoxycholate and Trasylol(25 KIU/ml) followed by two daily changes of 20 mM Tris-HCl buffer, pH 8.0, containing 0.5% taurocholate and Trasylol (25 KIU/ml). The dialysed sample was concentrated (20-30-fold) using a Centriconmicroconcentrator (Amicon). Samples were incubated for 3 hr at room temperature with an equal volume of a modified SDS-PAGE sample treating buffer containing 4% SDS prior to gel electrophoresis. This sample treatment was necessary to minimize the difficulties with SDSSPAGE caused by the concentrated nonionic detergent. Ultrapure reagents and deionized, double-distilled H,O were used in the following experiments. The SDS-PAGE separating gels were allowed to polymerize overnight at room temperature before being used, and thioglycolic acid (final 0.1 mM) was added to the upper gel running buffer in the SDS-PAGE. Reduced proteins separated by SDS-PAGE were transferred to a PVDF membrane using the same conditions as those described above in Western blot analysis except that 0.1 mM thioglycolic acid was included in the CAPS transfer buffer. After electroblotting, the membrane was washed, stained with Coomassie Brilliant Blue R-250, and destained as described by Matsudaira (1987). The stained protein bands were individually cut out with a clean razor. NH,-terminal amino acid sequencing of the stained protein was performed with a protein sequencer model 477A with a blot cartridge (Applied Biosystems, Foster City, CA) according to the manufacturer’s program, BLOTT- 1. The sequences obtained were compared with all reported sequences in several databases including the Gen Pept database (Release 74.0, 12/92).

S. VASILE et

422

As a control, the acid eluate from the control MOPC 195 variant immunoadsorbent column in the second round of immunoaffinity chromatography (see above) was treated in the same manner as the eluate from the SN8 immunoadsorbent column. A few very faint protein bands were detected and the amount of protein detected was not sufficient for protein sequencing.

al.

A

B

200-

m-

116 -

116-

97.4 -

97.4-

RESULTS

Detection of a heterodimeric antigen from human B cells by Western blotting and radioimmunoprecipitation BALL- 1, a human B leukemia cell line, expresses both mIgM-1, and mIgD-1. as determined by FACS analysis. An SN8 antigen preparation was isolated from BALL-l cells by detergent lysis and immunoaffinity chromatography (see Materials and Methods for details). The acid eluate from the SN8-column was analysed by Western blotting (Fig. 1). Under unreduced conditions, the antigen detected by SN8 had an approximate M, of 80 kDa (Fig. 1A). Under reduced conditions, SN8 reacted with a major component of approximately 36&39 kDa and a minor component of 34 kDa (Fig. 1B). The SN8-column eluate was radiolabeled with lZiI and subjected to immunoprecipitation and SDS-PAGE (Fig. 2, panel A). Under unreduced conditions, the SN8 immunoprecipitate (Fig. 2A, lane 1) migrated on gel electrophoresis as a diffuse major band of 73-85 kDa and another diffuse band of higher M, (probably aggregates), whereas no significant material was present in the immunoprecipitate with an isotype-matched murine control IgG (Fig. 2A, lane 2). Under reduced conditions, the SN8 immunoprecipitate showed three components of approximately 4447, 36-38 and 34 kDa (Fig. 2A, lane 3), whereas no bands were evident for the control IgG precipitate (Fig. 2A, lane 4). The 37 and 34 kDa components in Fig. 2A correspond to the major and minor components, respectively, detected by Western blotting (see Fig. 1B). The radioimmunoprecipitation and SDS-PAGE were also carried out using a ‘251-labeled antigen preparation from B PLL cells (Fig. 2, panel B); this antigen preparation was isolated from cell membrane glycoprotein mixtures as described previously (Okazaki et al.. 1993). Under unreduced conditions. the SN8 immunoprecipitate (Fig. 2B, lane 1) showed a single major component of approximately 80 kDa, whereas under reduced conditions, the SN8 immunoprecipitate showed two components of approximately 49 and 40 kDa (Fig. 2B, lane 3). The control IgG did not precipitate any significant component under either unreduced or reduced conditions (Fig. 2B, lanes 2 and 4). Thus, SN8 immunoprecipitated a disulfide-linked heterodimer, consisting of a 44-19 kDa (r chain) and a 36-40 kDa (fi chain) component, from both BALL-l and B PLL antigen preparations. In the case of the BALL-l antigen, SN8 immunoprecipitated an additional minor 34 kDa component. This component is probably disulfide-linked to the CIchain. This assumption is based on the facts that under unreduced conditions. the 34 kDa component was

66-

66-

Fig. 1. Western blot analysis of the SN8 antigen purified from BALL- 1 cells by immunoaffinity chromatography. Proteins recognized by SN8 were visualized on Kodak X-OMAT AR film using enhanced chemiluminescence (Amersham). The SN8 column eluate was examined on 8% SDS-polyacrylamide gels under nonreduced conditions (A) or after reduction with 0.1 M DTT (B) (see Materials and Methods for details). Arrows indicate SN8 antigen specific bands. BioRad M, marker proteins were used as references and their relative molecular masses are indicated in kDa.

not detected, but a minor component with a slightly lower M, than 80 kDa was detected in both Western blot analysis (Fig. 1A) and radioimmunoprecipitation (Fig. 2A, lane 1) of the BALL-l antigen preparation. The 34 kDa component is apparently an alternative form of the 3&40 kDa component since both share the epitope recognized by SN8. IdentiJication and churacterization of the human B29 and mb-1 proteins by amino acid sequence analysis In order to identify the protein detected by SN8, we determined the amino-terminal amino acid sequences of the individual components of the heterodimer. To this end, the SN8 antigen purified from BALL-l cells by immunoaffinity chromatography was concentrated, reduced and subjected to SDS-PAGE. The separated proteins were electroblotted to a PVDF membrane and the transferred proteins were detected by staining with

Human

B29 and mb-1 proteins

423

zoo11697.4e

66--

:

Fig. 2. Radioimmunoprecipitation of the SN8 antigen from a ‘2SI-labeled antigen preparation from BALL-l (panel A) and a ‘251-labeled B PLL antigen preparation (panel B). An isotype-matched murine IgG (IgGl-rc) was used as a control against SN8 (lanes 2 and 4 in panels A and B). The immunoprecipitates were unreduced (lanes 1 and 2 in panels A and B) or reduced with DTT (lanes 3 and 4 in both panels) before they were subjected to SDS-PAGE. Arrows depict specifically immunoprecipitated proteins which were visualized by autoradiography. M, marker proteins (BioRad) are indicated in kDa.

Coomassie Blue R-250 (Fig. 3). In a parallel experiment, the D chain component on the transfer membrane was detected by Western blot analysis. The 3638 kDa component detected by Coomassie Blue staining and Western blotting was observed as a diffuse band. In some experiments of SDS-PAGE followed by electroblotting, this protein band appeared to be composed of two closely situated protein bands. Therefore, this diffuse protein band on PVDF membranes was cut into two portions, the upper and lower portions, as indicated in Fig. 3. They were analysed separately by amino-terminal amino acid sequencing. An identical amino-terminal sequence was obtained for the first 14 amino acid residues for the two protein samples. In addition, arginine was identified at the 17th position for the lower band material (Fig. 4). No major amino acid residue was detected at position 17 in the upper band presumably because there was less protein in the upper band compared to the lower band (see Fig. 3). The amino-terminal sequence for the first 14 amino acid residues of the 3638 kDa p chain agreed completely with a portion (position 2942) of the deduced amino acid sequences from the human homologue of the murine B29 cDNA recently reported by Miiller et al. (1992) and Hashimoto et al. (1993). They deduced that amino acid residues for positions 15, 16 and 17 were half cystine, serine and arginine, respectively. Upon comparing their deduced

amino acid sequence with the amino acid sequence of murine B29 protein, Miiller et al. (1992) suggested that the human B29 protein possesses a 30-amino acid leader sequence and an extracellular domain of 129 amino acids whose amino-terminal sequence is Ser-Glu-Asp-ArgTyr-. On the other hand, Hashimoto et al. (1993) proposed that the human B29 protein possesses a 25 amino acid leader sequence and an extracellular domain of 134 amino acids whose amino-terminal sequence is Val-Pro-Ala-Ala-Arg-Ser-Glu-Asp-Arg-Tyr-. However, our results clearly show that the amino-terminal sequence of the extracellular domain of the human homologue of the murine B29 protein is Ala-Arg-SerGlu-Asp-Arg-Tyrindicating that the human B29 homologue has a 28-amino acid leader sequence and an extracellular domain of 13 1 amino acids. In Fig. 4, the amino-terminal sequence of the human homologue of the murine B29 protein (hitherto called the human B29 protein) is compared with that of the murine B29 protein (Hermanson et al., 1988; Hombach et al., 1990a). Concerning the c( chain component of the heterodimer isolated from BALL-l, three closely situated protein bands (indicated by arrows in Fig. 3) were detected by Coomassie staining at the area of the cc chain (4448 kDa) after the reduced BALL-l antigen preparation was subjected to SDS-PAGE followed by electroblotting to a PVDF membrane. These bands were cut

S.VASILEet al.

424

4

/3-tubulin

presented in Fig. 4. The comparison shows that the majority of the conserved amino acid residues are hydrophobic amino acids which include leucine I, 10, 14, proline 7, valine 12 and alanine 18. Such conservation of hydrophobic amino acid residues is not observed in the amino-termini of the human and mouse B29 proteins (Fig. 4). The determined amino-terminal amino acid sequence of the human mb-1 protein is consistent with the predicted length (32 amino acid residues) of the leader sequence and the predicted amino-terminal amino acid sequence of the human mb-1 gene product (Ha et al., 1992; Yu and Chang, 1992). ~dent~~catio~ of a protein copur~~ed with the mb-I -B29 dimer as a ~-t~bu~in-fake protein by amino acid sequence ana/Jsis

Fig. 3. Coomassie Blue visualization of the immunoaffinity purified SN8 antigen. The SNX antigen was purified from a detergent lysate of BALL-l cells by immunoa~nity chromatography. The sample was concentrated, reduced with DTT. subjected to SDS-PAGE, and transferred to a PVDF membrane by electroblotting (see Materials and Methods for details). The transferred proteins were visualized by staining with Coomassie Blue R-250. The 44-48 kDa proteins indicated by the upper three arrows were determined to possess an identical amino-terminal amino acid sequence which corresponds to the sequence of the human mb- 1 protein. The diffuse 36-38 kDa B29 protein band was divided into two portions as indicated in the figure and separately analysed by amino acid sequencing; an identical amino-te~inal amino acid sequence was obtained from the two samples. The arrow head indicates the protein band containing a ~-tubu~in-like protein as determined by amino-terminal amino acid sequence analysis. out and analysed separately by amino acid sequencing. The three samples showed an identical amino-terminal sequence (Fig. 4), which was found to correspond to a portion (position 33-52) of the deduced amino acid sequence from a recently reported nucleotide sequence of human mb-I cDNA clones (Ha et al., 1992; Yu and Chang, 1992). For a comparison, the amino-terminal amino acid sequence of the murine mb-I protein (Sakaguchi et al., 1988; Hombach e7t uf., 1990n) is also

The three protein bands that were detected in the area above the mb-1 protein bands, i.e. between 50 and 70 kDa (Fig. 3) were also subjected to amino acid sequence analysis; among these bands, the middle band of approximately 58 kDa was the most intense as indicated by an arrow head in Fig. 3. Only this sample yielded a significant amino acid sequence, which was Met-Arg-Glu-Ile-Val-His-Ile-Gln-Ala-Gly-Gin. This sequence completely agrees with the amino-terminal amino acid sequence of human a,-tubulin as revealed by a computer search. To investigate whether the binding of the fl-tubulin-like protein to the SN8 immunoadsorbent column is due to specific interaction, the following control experiment was performed: an acid eluate from the control MOPC immunoadsorbent in the second round of immunoaffinity chromatography (see Materials and Methods} was dialysed and concentrated in the same manner as the SN8-immunoadsorbent acid eluate in which the ~-tubulin-like protein and the mb-1 and I329 proteins were found. The result of SDS-PAGE of the acid eluate from the MOPC immunoadsorbent is shown in Fig. 5. Only very faint protein bands were detected on the areas of the PVDF membrane which may correspond to the mb-1 protein and perhaps the p-tubulin-like protein. The result sugests that the fi-tubulin-like protein bound to the SN8 immunoadsorbent specifically, probably via the mb-l-B29 dimer. Amino acid sequence homology between the MT-binding domains of MAPS and the mb-1 protein The co-purification of a ~-tubulin-like protein with the mb-l-B29 antigen led us to examine the mb-l-B29 protein sequences for potential MT-binding domains. We found that there is a strong amino acid sequence homology between the MT-binding domains of certain human MAPS and an intracellular segment of the human mb-1 protein (Fig. 6). DISCUSSION Previously, we generated three mAbs, termed SN8, SN8a and SN8b, by immunizing mice with a glycoprotein antigen preparation isolated from B PLL cells (Okazaki et ul.. 1993). The antigen preparation was

Human 5

B29 and mb-1 proteins

HnB29 :

Ala Arg m

Glu m

10 Arg Tyr Arg ban Pro Lys m

MoB29:

-

Ser m

~eu Pro Leu m

HUIIB-1: u

-

m

Trp I4et His

5 dye Val PLp Ala w

m

Phe Gln ELy m

425 15 Ala ND

ND

Pro Cys Ser

Arg Gln

15 10 &!J Hat yal Ser i&u CilyiZ,lu ASP ti

20 His Phe

Fig. 4. Amino-terminal amino acid sequences of the human B29 and mb-I proteins. For a comparison, the amino-terminal sequences of the mouse B29 and mb-I proteins are shown after alignment to the corresponding human homologues to maximize the homology. Identical amino acid residues between the corresponding human and mouse proteins are underlined. Shifts introduced in the sequence to maximize alignment are designated by a dash.

isolated from the cell membrane glycoproteins using our previously developed procedure (Seon et al., 1981). Analyses of the reactivities of these mAbs with various human leukemia-lymphoma specimens showed that each of them reacted with an extracellular epitope of a cell membrane antigen on human B leukemialymphoma cells, particularly with B PLL, B nonHodgkin’s lymphoma and B ALL cells (Okazaki et al.,

116 97.4

31

21.5 Fig. 5. SDS-PAGE analysis of the acid eluate from the control MOPC column. The sample was concentrated, reduced with DTT, subjected to SDS-PAGE, and transferred to a PVDF membrane by electroblotting as described in the legend to Fig. 3. The transferred proteins were visualized by staining with Coomassie Blue R-250.

1993). These mAbs showed no significant reactivity with normal human peripheral blood T cells, granulocytes, monocytes, erythrocytes or platelets, but did react with B cells. In the present study, the heterodimeric antigen defined by SN8 was identified as the human mb-l-B29 heterodimer. Furthermore, SN8 was determined to react with the B29 component of the dimer. Recently, the putative human homologues of the mouse B29 and mb-1 proteins were reported for human B cells to exist as 3740 and 47-50 kDa components (van Noesel et af., 1991; Mason et al., 1991; Nakamura et al., 1992; Clark et al., 1992; Leprince et al., 1992). In this study, we detected a heterodimeric antigen composed of a 3640 and a 4449 kDa component in the antigen preparations from B PLL cells and BALL-l leukemia cell line by using radioimmunoprecipitation and Western blot analysis. An additional minor component of 34 kDa was detected in the reduced antigen from BALL- 1. To further characterize the SN8 antigen, we isolated the non-radiolabeled human homologues and chemically identified them as the human B29 and mb-1 proteins by determining the amino-terminal amino acid sequences of the individual proteins. The results in Figs 3 and 4 demonstrate that the human mb-I protein and perhaps also the human B29 protein are molecularly heterogeneous. Campbell et al. (1991) detected a similar heterogeneity of the murine mb-1 protein by SDS-PAGE of the radiolabeled protein. They attributed this heterogeneity to the differential glycosylation of the protein. In a recent preliminary study, BALL-l cells were surface labeled with ‘251. A portion of the ‘251-labeled BALL- 1 cells was lysed with 1% digitonin and the lysate was subjected to immunoprecipitation using SN8 or goat anti-human IgM antiserum. The result indicated that the mb-l-B29 heterodimer on BALL-l cells was associated with mIg. In the present study, a protein which was co-purified with the mb-l-B29 heterodimer was identified as a p-tubulin-like protein by determination of the aminoterminal amino acid sequence. CI- and p-tubulin are the structural proteins of MTs which are important in a variety of dynamic cellular events as well as in maintaining many stable cellular structures (Chapin and Bulinski,

426

s. VASILE

HuMB-1

Thr

MT-binding

domain

Tyr

Ala Gln

rt al. 190 185 Asn Ile ... Gly ... Asp )QJ Gin Leu Glu

180 Gin Asp Val Gly Ser

Leu -

Ala

Leu Asp Asn

Lys Val Gly Ser

X, X, . .. . .

His

m

Pro Gly Gly

Fig. 6. Comparison of the human mb-I protein with the MT-binding domains of three human MAPS: MAP 2, tau and MAP 4. The deduced amino acid sequence of the human mb-I protein was taken from Yu and Chang (1992) and Ha a ~1. (1992). but numbered as determined in this report (see Fig. 4) for the mature mb-I protein. The consensus sequence of the MT-binding domains of MAP 2, tau and MAP 4 was derived from Chapin and Bulinski (1992) and shown in the figure. The .x, and xl in the sequence represent variable amino acid residues: X, is alanine. isoleucine and valine. respectively, in MAP 2, tau and MAP 4, whereas .Y?is histidine, threonine and glycine, respectively. in the three proteins. A gap was introduced in the mb-I protein sequence to maximize the homology.

1992; Luduena

rt

al.,

1992). MTs

MAPS in Co;

are

defined

as

an operational definition includes proteins that co-purify with MTs (Chapin and Bulinski, 1992; Luduena et al., 1992). A number of MAPS, including the assembly-promoting MAPS, have been reported. Each of the assemblypromoting MAPS possesses MT-binding domains, and the amino acid sequences of these domains were shown to be highly conserved among three MAPS, termed MAP 2, MAP 4 and tau [reviewed in Chapin and Bulinski (1992)]. We compared the amino acid sequences of the MT-binding domains with those of the human mb- 1 and B29 proteins, and found that there is a strong homology in the sequence between the MT-binding domains and an intracellular segment of the mb- 1 protein (Fig. 6). The sequence motif found in the human mb-1 protein and human MT-binding domain is less distinct in the mouse mb-I protein. In the mouse mb-I protein, three of the nine amino acid residues found in the sequence motif of the human mb-1 protein are replaced. i.e. Ser 183, Asn 185 and Val 189 are replaced with Asn, His and Ala, respectively. It is interesting to note that if the comparison is made between the human and mouse mb-1 proteins for the region containing the sequence motif, e.g. residue 160 to the carboxy termini (i.e. residue 194 for the mature human mb-1 protein), amino acid sequence differences are found only within the above mentioned motif. When the sequence homology is taken together with the fact that a fl-tubulin-like protein was co-purified with the mb-l-B29 antigen. the present results suggest that the mb-l-B29 heterodimer may be associated with MTs, via the mb-I protein, in B cells. However, further studies are needed to establish the specific association between the heterodimer and MTs, and to understand the nature of the association. It is worthy to note that SN8 was the only mAb that reacted with an extracellular epitope of the human mb-l-B29 heterodimer at the recent Fifth International Conference on Human Leukocyte Differentiation Antigens (Schlossman et al., 1994). Furthermore, SN8 induces signal transduction in normal B cells (van Kooten et al., 1994). proteins

associated

with

Acknowledgements-We thank Mr M. Garfield, MS P. Tingue and Mrs H. Tsai for their technical assistance and Mrs S. Sabadasz for her help in the preparation of the manuscript.

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