b-lymphocyte development)

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 676-680, January 1991 Immunology Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen...
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Proc. Nati. Acad. Sci. USA Vol. 88, pp. 676-680, January 1991 Immunology

Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3 (ectopeptldases/angiotensins/B-lymphocyte development)

Q. Wu*t, L. LI*, M. D. COOPER*t, M. PIERRES§, AND J. P. GORVEL¶ *Division of Developmental and Clinical Immunology, Departments of Pediatrics, Medicine, and Microbiology, and The Comprehensive Cancer Center, University of Alabama at Birmingham, and the tHoward Hughes Medical Institute, Birmingham, AL 35294; §Center d'Immunologie, Institut National de la Sante et de la Recherche M6dicale, Centre National de la Recherche Scientifique Case 906, 13288 Marseille Cedex 9, France; and '1 Meyerhofstrasse, Postfach 10.2209, D-6900 Heidelberg, Federal Republic of Germany

Contributed by Max D. Cooper, October 22, 1990

The predicted amino acid sequence of the ABSTRACT cDNA encoding the murine B-lymphocyte differentiation antigen BP-1/6C3 suggested that it is a member of the zincdependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [microsomal aminopeptidase; a-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2]. In the present studies, we examined the enzymatic activity of this antigen. From brush border preparations of the small intestine, a rich source of many endopeptidases and exopeptidases, the BP-1 antibody selectively removed aminopeptidase A [APA; L-a-aspartyl(L-a-glutamyl)-peptide hydrolase, EC 3.4.11.7] activity. The APA activity of a panel of cell lines correlated in linear fashion with cell-surface levels of the BP-1/6C3 antigen. APA activit was demonstrated for the BP-1/6C3 antigen immunopurified from the pre-B-cell membrane. This activity was enhanced by alkaline earth metals such as Ca2' and was abrogated by amastatin and angiotensin, which are known competitive inhibitors of APA. The data indicate that the murine BP-1/6C3 antigen is active APA, an enzyme that catalyzes specifically the removal of unsubstituted, N-terminal glutamic acid and aspartic acid residues from peptides.

aminopeptidase N [APN; microsomal aminopeptidase; a-aminoacyl-peptide hydrolase (microsomal) EC 3.4.11.2]. Four distinctive types of aminopeptidases have been reported (12). These include APN, which has a relatively broad specificity acting on peptides with an N-terminal neutral amino acid; aminopeptidase A [APA; u-a-aspartyl(L-a-glutamyl)-peptide hydrolase, EC 3.4.11.7], acting on peptides with N-terminal acidic amino acids; aminopeptidase P (aminoacrylprolyl-peptide hydrolase, EC 3.4.11.9) and aminopeptidase W, which hydrolyze peptides in which the penultimate amino acid is a proline or a tryptophan. In the present studies, we have obtained evidence that the BP-1/6C3-reactive molecule exhibits APA activity.

MATERIALS AND METH1ODS Cells and Antibodies. Cell lines (see Table 2; refs. 3, 4, and 7) were maintained in RPMI 1640 medium (GIBCO) supplemented with 10%o fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 ,ug/ml), 2 mM glutamine, and 50 ,uM 2-mercaptoethanol. BP-1 antibody is a mouse IgG2a monoclonal alloantibody (2). The control 7108.2 antibody is a mouse IgG2a antibody with reactivity against human major histocompatibility complex class I antigen (3). Fluoresceinconjugated goat anti-mouse IgG2a was purchased from Southern Biotechnology Associates (Birmingham, AL). Flow Cytometric Analysis. Cell (1 x 106) were incubated on ice for 15 min in an excess of either BP-1 antibody or the 7102.8 control antibody. After two washes with cold phosphate-buffered saline (PBS) containing 5% FCS and 0.1% sodium azide (NaN3), the cells were incubated for 15 min on ice with fluorescein-conjugated goat anti-mouse IgG2a. To exclude dead cells from analysis, after two washes with PBS/5%FCS/0.1% NaN3 the cells were resuspended in the same buffer containing propidium iodide (10 ,ug) and were analyzed with a FACScan (Becton Dickinson). The mean fluorescence intensity of BP-1-positive cells was determined relative to the background fluorescence intensity of cells preincubated with the control antibody. Membrane Preparations. Plasma membrane samples of the cell lines were prepared as described (11). Small intestinal membrane vesicles were prepared by a procedure involving addition of CaC12 and differential centrifugation of homogenized small intestinal mucosa (13). Immunoprecipitation and Immunodepletion. The methods of solid-phase immunoprecipitation and SDS/PAGE have been described (2). In immunodepletion experiments, AffiGel-10 beads (Bio-Rad) coated with rabbit anti-m1ouse

The murine BP-1/6C3 antigen was originally identified by BP-1 and 6C3 monoclonal antibodies on normal and transformed pre-B and immature B lymphocytes as a homodimeric, phosphorylated cell-surface glycoprotein with 140kDa subunits (1-3). Expression of this antigen was subsequently shown on some bone marrow-derived stromal cell lines, brush borders of the proximal renal tubules and small iptestinal enterocytes, and a subpopulation of thymus cortical epithelial cells (refs. 4-6; unpublished results). While its broad tissue distribution implied diverse biological function, studies of the BP-1/6C3 antigen thus far have been focused mainly on its possible role in B lymphocyte development. The BP-1/6C3 molecule is expressed on pre-B and immature B-lineage cells in the bone marrow (2, 3, 7), and its expression on stromal cell lines correlates with their ability to support pre-B-cell growth (4). Moreover, interleukin 7 (IL-7) preferentially induces BP-1/6C3 expression together with pre-B-cell proliferation (6, 8) and up-regulated expression of the BP-1/6C3 antigen, which is often seen on transformed pre-B cells (1, 2, 9, 10). In an effort to define the structure and function of this interesting cell-surface molecule, we purified the antigen and cloned its cDNA from an Abelson murine leukemia virustransformed pre-B-cell line (11). The amino acid sequence predicted from the cDNA sequence suggested that the murine BP-1/6C3 antigen is a member of the zinc-dependent metalloprotease family, possibly an aminopeptidase related to

Abbreviations: IL-7, interleukin 7; APA, -N, and -B, aminopeptidases A, N, and B. tPresent address: The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Cahada.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Immunology: Wu et al.

Proc. Natl. Acad. Sci. USA 88 (1991) Reduced

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