Purification, composition, and structure of macrophage adhesion molecule

Macrophage adhesion molecule (MAM) is a surface heterodimer consisting of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). MAM, which is the guinea pig analogue of Mo1 and Mac-1, was purified from detergent lysates of peritoneal neutrophils by lentil lectin chromatography and M2-antibody chromatography. The pure heterodimer molecule was dissociated by acidic conditions (pH 3.5), and MAM-alpha and MAM-beta were separated by M7-antibody chromatography. MAM-beta is an approximately 640 amino acid residue polypeptide with exceptionally high cysteine content. At 7.2 residues per 100 amino acids, Cys/2 of MAM-beta is more than 3 times the mean for 200 purified proteins. Reactivity with six beta-subunit-specific monoclonal antibodies recognizing at least four epitopes demonstrated that intrapeptide disulfide bonds are required to maintain the structure of MAM-beta. All six antibodies failed to react when MAM-beta was treated with reducing agents. MAM-beta is 18% carbohydrate; the major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid. MAM-beta is estimated to contain five to six N-linked carbohydrate units. MAM-alpha is an approximately 1100-residue polypeptide with lower Cys/2 content (2.0 residues per 100 amino acid residues). MAM-alpha is 21% carbohydrate. The major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid; the mannose content is higher in MAM-alpha than MAM-beta. MAM-alpha is estimated to contain 12 N-linked carbohydrate units.     

Structure and function of macrophage adhesion molecule examined by epitope mapping

Macrophage adhesion molecule (MAM), a member of the integrin superfamily of heterodimer membrane molecules with adhesive properties, is the guinea pig counterpart of human Mo1 (CD11b/CD18). Earlier work showed that MAM is synthesized as monomeric precursor glycopeptides that assemble to form the heterodimer. The heterodimer and monomer glycopeptides are characterized through the use of twelve mAb in immunoprecipitation, immunoblotting, binding assays, and a quantitative cell adhesion assay. Seven topographic regions are identified, two of which are shown to be critical for adhesion. One adhesion-related topographic region, the M2/M4 region, is on the alpha-subunit, and the other, the M8/M15 region, is on the beta-subunit. Both adhesion-related epitopic regions are not detectable on monomeric glycopeptides but are generated by conformational change on heterodimer formation. It is hypothesized that these structure-function relationships have general applicability to integrin molecules.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

A novel guinea pig macrophage-specific polymorphic molecule. II. Biochemical analysis of the polymorphism

We have identified a macrophage-specific molecule, termed gp98, which has a m.w. of 98,000, is encoded by a gene not linked to the guinea pig lymphocyte antigen complex, is highly immunogenic, and displays a serologic polymorphism among several inbred guinea pig strains. The gp98 molecule was biochemically analyzed to identify a basis for the serologically detected polymorphism. The molecule was demonstrated to be a glycoprotein containing N-linked oligosaccharides. The strain 2 serologic variant, gp98-2, migrated with an apparent m.w. approximately 2500 more than did the strain 13 variant gp98-13. This differential migration was observed in a (strain 2 X strain 13) F1 animal, and persisted after neuraminidase and endoglycosidase F treatment, and after reduction. Trypsin and endoproteinase Lys-C digestion localized the biochemical basis of the polymorphism to the peptide portion of the molecule. Biochemical analysis of the gp98 molecules from five different inbred strains indicated that only two biochemical variants correlating with the serologic variants existed among the five strains.     

Regulation of synthesis of macrophage adhesion molecule, a heterodimeric membrane glycoprotein

Macrophage adhesion molecule is a surface molecule of guinea pig macrophages and neutrophils. It is the counterpart of mouse Mac-1 and human CD11b/CD18 (Mol/OKM-1/Mac-1/Leu-CAM) and is member of a family of heterodimer glycoproteins with a common beta-subunit. Macrophage adhesion molecule is a prevalent molecule in nonactivated macrophages, but it is dramatically decreased in macrophages activated in vivo. The experimental system of activated vs nonactivated guinea pig peritoneal macrophages was used to examine the mechanisms that down-regulate synthesis of this heterodimer molecule. [35S]Methionine labeling of nonactivated macrophages and chase incubation revealed that synthesis involves separate translation of the alpha- and beta-glycopeptides of "high mannose"-containing monomeric precursors, then refolding/assembly to form a heterodimer, and, finally, a maturation process that includes conversion of carbohydrate to "complex" units. Two lines of evidence demonstrate that down-regulation in activated macrophages occurs via restriction of the alpha-species. First, pre-beta is detected at 3 h only in activated macrophages. Second, the amount of newly translated pre-alpha averaged 16% in activated macrophages relative to nonactivated macrophages, which is close to the value of 12% for the mature heterodimer. The amount of newly translated pre-beta averaged 62%. These findings identify the regulatory step as a restriction of the alpha-species at, or before, translation. A model is proposed to explain regulation of synthesis of heterodimer membrane glycoproteins.     

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.     

Further Studies on Guinea Pig Z-1 Antigen That Is Involved in Phagocytosis of Zymosan by Macrophages: Cell Type Distribution of the Antigen and Cross-Reactivity of Anti-Z-1 with Human CR3

We have previously identified a heterodimer molecule, Z-1, on guinea pig peritoneal macrophages (Møs) by the newly prepared monoclonal antibody, anti-Z-1, and Z-1 has been assumed to be the complement receptor type three (CR3) in this species. To clarify this assumption, the cell type distribution of the antigen in guinea pig and the cross-reactivity of anti-Z-1 with other species were analyzed. It was demonstrated that Z-1 was expressed on peritoneal Møs, pulmonary Møs, peritoneal polymorphonuclear leukocytes (PMN), peripheral neutrophils, and some subpopulations of spleen cells and of bone marrow cells, but not on erythrocytes, circulating lymphocytes, and lymphocytes in both spleen and bone marrow in detectable amounts. Thus the expression of Z-1 seems to be restricted to phagocytes as is CR3 of other species. Furthermore, it was found that anti-Z-1 bound with peripheral neutrophils from human, horse and goat and HL-60 cells differentiated into monocytes. Any cross-reactivity of the antibody was not detected with neutrophils from rabbit, cow, sheep and dog and nondifferentiated HL-60 cells. Human Z-1 was indistinguishable from human CR3, since both were the heterodimer consisting of α chain of 170 kDa (pI = 6.6-7.2) noncovalently associated with β chain of 100 kDa (pI = 5.6-6.7). In addition, human CR3 in detergent-lysate of neutrophils was completely adsorbed with anti-Z-1 F(ab')2-Sepharose. These findings indicate that guinea pig Z-1 shares an antigenic determinant with human CR3. It thus seems to be possible that Z-1 may function as CR3 in guinea pigs.     

Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin

We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions.     

Some structural and functional aspects of the cell adhesion molecule uvomorulin

The cell adhesion molecule uvomorulin (UM) was analysed by comparing antisera produced against the whole molecule (gp123) with antisera made against fragments of UM. Of the proteins recognized by different anti-UM antisera (molecular weights of 123, 102, 92 and 84 kDa), the 102 kDa molecule is not derived from gp123. The 102 kDa molecule is not glycosylated and is also different from gp123 by peptide map analysis. However, rabbit antisera raised against the purified 102 kDa protein interfered with the aggregation of embryonal carcinoma (EC) cells. Also, a monoclonal antibody selected to interfere with EC cell aggregation recognized the 102 kDa molecule as well as gp123. Thus, the functional site of cell adhesion seems not to be mediated by sugar residues. Experimental evidence is provided suggesting that UM is not only involved in the compaction of preimplantation embryos but seems to be an ubiquitous cell adhesion molecule regulating epithelial cell adhesion mechanisms.     

The Kurloff cell in estrogenized guinea pigs as a CT7+ 8BE6- CT6- MR-1- CT10- IgM- lymphocyte with natural killer activity.

The relationship of the Kurloff cell (KC), guinea pig blood mononuclear cell with natural killer (NK) activity, to a known cell lineage was established. Using indirect immunoperoxidase staining and flow cytometric analysis, numerous monoclonal antibodies directed against guinea pig macrophage antigen, Ia antigen or different T lymphocyte markers and a polyclonal anti-IgM serum were tested in unimmunized estrogenized animals. We excluded any relationship between KC and the monocytic macrophage lineage (MR-1-) and between KC and the B lymphocyte lineage (CT10- and IgM-). The KC immunophenotype was pan T CT7 positive but 8BE6 (mature thymocyte) and CT6 (cytotoxic suppressor T lymphocyte) negative. Since KC displays an NK activity, this cell may be classified among the NK effector cells exhibiting some T lymphocyte markers.     

Expression in yeast of a cDNA clone encoding a transmembrane glycoprotein gp41 fragment (a.a. 591-642) bearing the major immunodominant domain of human immunodeficiency virus.

A cDNA clone corresponding to the gp41 gene fragment nucl. 7573-7730 of the human immunodeficiency virus type 1 (HIV-1) was selected from a random HIV-1 genomic library expressed in yeast. This clone encodes a 52-residue long peptide (amino acid (a.a.)) 591-642) bearing the major immunodominant domain (a.a. 598-609) of the HIV-1 transmembrane glycoprotein gp41. Expression of the recombinant peptide pSE-env591-642 was driven by the alpha-mating factor leader sequence contained in a plasmid pSE-x allowing the synthesis and secretion of foreign gene product in Saccharomyces cerevisiae. Time-course analysis of the secretion into culture medium revealed an optimal production of the glycoprotein fragment at 28-30 h with no observable cytotoxicity. The secreted peptide is highly glycosylated with NH2-terminal heterogeneity probably due to different post-translational modifications. The secreted peptide shows an extreme antigenicity since in ELISA assays, as few as 5 microliters/well of crude supernatant are sufficient to obtain a strong detection by monoclonal antibodies or by 100% of sera from HIV-infected individuals. The purified glycopeptide pSE-env591-642 binds to a monoclonal antibody directed against the immunodominant epitope (a.a. 603-609) with an affinity similar to that of the complete glycoprotein gp160 (Kd values within the 10(-10) M range) and with a 100-fold higher affinity than that of a linear peptide fragment SP-env584-609. These results indicate that overexpression in yeast can efficiently provide an abundant source of highly antigenic gp41 protein fragment pSE-env591-642 which retains the antigenic properties of the native gp160 protein. Such a recombinant peptide should therefore be considered as a good candidate for antigen in HIV detection tests.     

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.     

Decrease of the major surface glycoprotein gp160 in activated macrophages

The quantity of surface-radioiodinated gp160, the previously described trypsin- and plasmin-sensitive surface glycoprotein of guinea pig macrophages was reduced 70% in macrophages activated in vivo in comparison to elicited macrophages. The reduction of gp160 was also detected by Coomassie blue staining, demonstrating that the absolute number of gp160 molecules is lower in activated macrophages. Gp160 was present on activated macrophages as single-chain intact molecules; no proteolytically cleaved gp160 was detected. Biosynthesis of gp160 was compared for activated and elicited macrophages by culturing with [³⁵S]methionine. Whereas biosynthetically labeled gp160 was readily detected in elicited macrophages, negligible [³⁵S]methionine-labeled gp160 was detected in parallel cultures of activated macrophages, suggesting that a major cause of decreased surface gp160 on activated macrophages lies at the level of synthesis.     

Mediation of cell-cell adhesion by the altered contact site A glycoprotein expressed in modB mutants of Dictyostelium discoideum

In Dictyostelium discoideum, a surface glycoprotein with Mr 80,000 (gp80) has been found to mediate the EDTA-resistant contact sites A at the aggregation stage of development. To evaluate the role of the carbohydrate moiety in cell-cell adhesion, we have examined the accumulation and activity of an altered gp80 molecule in two glycosylation (modB) mutants. Both mutants synthesize an altered gp80 of lower molecular size. This modB-gp80 can be detected by the monoclonal antibody 80L5C4, which is capable of blocking cell-cell adhesion (C. -H. Siu, T. Y. Lam, and A. Choi, (1985) J. Biol. Chem. 260, 16,030-16,036). The mutant cells exhibit both EDTA-sensitive and EDTA-resistant types of cell-cell binding, though to a lesser extent than that of the parental strain, and the EDTA-resistant binding sites are blocked in the presence of 80L5C4 Fab. Mutant cells can also bind Covaspheres conjugated with gp80. These results suggest that the modB-gp80 protein still retains the domain essential for its cell binding activity and the carbohydrate moiety affected by the modB mutation is not directly involved in cell-cell adhesion.     

Inhibition of cell-cell binding at the aggregation stage of Dictyostelium discoideum development by monoclonal antibodies directed against an 80,000-dalton surface glycoprotein

Monoclonal antibodies were prepared against a putative cell-cell adhesion molecule, a surface glycoprotein with an apparent Mr of 80,000 (gp80), from Dictyostelium discoideum. Seven monoclonal antibodies directed against gp80 were characterized and found to fall into three distinct classes. Class I consisted of one monoclonal antibody, is monospecific for gp80, and probably recognizes the peptide portion of the molecule. This class was capable of blocking the EDTA-resistant contact sites effectively. Class II recognized the carbohydrate moiety of gp80 and cross-reacted with a large number of glycoproteins. These monoclonal antibodies partially inhibited cell reassociation. Class III recognized gp80 and one other glycoprotein of Mr 95,000. This class had no effect on cell-cell binding. The class I monoclonal antibody was most potent in inhibiting cell reassociation at the aggregation stage of development. Its effect decreased drastically as development progressed and became negligible by the culmination stage. These observations are consistent with a direct role of gp80 in cell-cell binding and suggest a transient function for gp80 at the aggregation stage.     

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.     

Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines

We have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. We now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bands are closely related polypeptides.     

The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu

The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPmu belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTPmu. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPmu ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTPmu ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTPmu at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.     

The identification, characterization, and distribution of guinea pig CCR4 and epitope mapping of a blocking antibody.

Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon gamma. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.