Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.     

Monoclonal antibody 45-2D9 recognizes a cell surface glycoprotein on a human c-Ha-ras transformed cell line (45-342) and a shared epitope on human tumors

A monoclonal antibody (45-2D9) produced after immunization of BALB/c mice with the c-Ha-ras NIH 3T3 tertiary transfectant (45-342) recognized a determinant expressed by the primary, three of three secondary, and one of three tertiary transfectants, but not by NIH 3T3 cells. The determinant was present on the cell surface and was distinct from murine leukemia virus gp70 by absorption studies. Biosynthetic labeling and immunoprecipitation studies with [35S]methionine and [3H]glucosamine demonstrated that 45-2D9 recognizes a 74,000 Mr glycoprotein with minor bands of 90,000 and 180,000 Mr on SDS-PAGE. Pulse chase studies demonstrated a 68,000 Mr precursor molecule that incorporated only [35S]methionine. The distribution of the epitope recognized by 45-2D9 was assessed by immunoperoxidase staining. The antigen was not detected on 10 primary and metastatic murine tumors or 11 transformed murine cell lines. However, a variety of surgically excised human tumors demonstrated intense staining, whereas staining of normal tissues was minimal or not detectable. Thus a human oncogene-transfected cell can express a new cell surface determinant apparently unrelated to the oncogene product, which is also selectively expressed by human tumors.     

Biosynthesis and turnover of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus

Antiserum was raised in rabbits against a bile canalicular glycoprotein of Mr = 110,000 purified to homogeneity from of rat liver. The antisera specifically immunoprecipitated a Mr = 110,000 polypeptide from hepatocytes metabolically labeled with [35S]methionine. When hepatocytes in primary culture were incubated with tunicamycin before labeling with [35S]methionine in the presence of tunicamycin, the major polypeptide immunoprecipitated by the specific antiserum from Triton X-100 extracts of cells had a molecular weight of 59,000. Enzymatic removal of N-linked carbohydrates from the Mr = 110,000 glycoprotein by N-glycanase digestion also yielded a polypeptide with minimum Mr = 59,000. In pulse-chase experiments using [35S]methionine, the Mr = 110,000 protein detected by the specific antisera first appears as Mr = 85,000 and 75,000 intermediate species which are endoglycosidase H sensitive. The Mr = 85,000 intermediate form is lost first with time followed by the Mr = 75,000 form giving rise to the Mr = 110,000 form that is endoglycosidase H resistant. Neuraminidase digestion of the Mr = 110,000 form generated an Mr 85,000 form but with a different carbohydrate structure than the intermediate Mr 85,000 form detected in the pulse-chase experiments. The time required to accomplish the processing of the Mr = 85,000 and 75,000 forms is relatively slow. Finally, the terminal sugars are added and the mature Mr = 110,000 glycoprotein is rapidly transported to the cell surface. A minimum time of 90 min is required for the Mr = 110,000 bile canalicular glycoprotein to be synthesized, processed, and reach the cell surface which is long relative to the time required (10 min) for another domain-specific protein, the receptor for asialoglycoproteins, to reach the sinusoidal surface. The Mr = 110,000 bile canalicular glycoprotein turns over in the bile canalicular domain with a half-life of 43 h while the asialoglycoprotein receptor turns over in the sinusoidal domain with a half-life of 23 h.     

Coculture of mouse IL-3-dependent mast cells with 3T3 fibroblasts stimulates synthesis of globopentaosylceramide (Forssman glycolipid) by fibroblasts and surface expression on both populations

Mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mouse 3T3 fibroblasts, cultured separately or together, were examined for their cell surface expression and biosynthesis of globopentaosylceramide, a marker of the mouse serosal mast cell. As assessed by flow cytometric analysis, BMMC cultured for up to 7 wk in 50% WEHI 3-conditioned medium containing IL-3 did not bind the B1.1 anti-globopentaosylceramide mAb (six experiments). A total of 10 +/- 4% (mean +/- SD, three experiments) of 3T3 fibroblasts that had reached confluence in medium without IL-3 bound B1.1 antibody and, after an additional approximately 28 days of culture in that medium or in 50% WEHI 3-conditioned medium, 12 +/- 3% (mean +/- SD, five experiments) and 16 +/- 7% (mean +/- SD, three experiments) of the cells, respectively, bound the antibody. After coculture of BMMC and confluent 3T3 fibroblasts for 28 days in 50% WEHI 3-conditioned medium, followed by dispersal and purification of the cells, 92 +/- 18% of the mast cells and 92 +/- 16% (mean +/- SD, seven experiments) of the fibroblasts were B1.1+. Whereas the increase in the expression of the epitope bound by B1.1 antibody on fibroblasts was noted by day 14 of coculture, expression of the epitope on mast cells did not occur until day 21 (three experiments). Biosynthesis of globopentaosylceramide was assessed by intrinsic radiolabeling of each cell population and identification of the extracted neutral glycosphingolipids by TLC and autoradiography. Synthesis of globopentaosylceramide was not detected in extracts of 9 x 10(6) BMMC, 1 x 10(6) confluent 3T3 fibroblasts cultured alone for 28 days, or 9 x 10(6) mast cells purified from 28-day cocultures but was readily detected in extracts of 3 x 10(5) fibroblasts purified from the same cocultures. These findings indicate that BMMC stimulate an increase in the synthesis and expression of globopentaosylceramide on 3T3 fibroblasts and suggest that the subsequent appearance of this neutral glycosphingolipid on the surface of the mast cells is due to its secretion by fibroblasts and adsorption to the mast cell surface. Thus, the interactions between mast cells and fibroblasts during coculture alter the biochemical and Ag phenotypes of both populations.     

Lysosome-associated membrane proteins: characterization of LAMP-1 of macrophage P388 and mouse embryo 3T3 cultured cells

Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, has been identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anit-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromatography with 1D4B monoclonal antibody, yielding a homogeneous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the two purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [35S]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent Mr 92,000, and then converted to heterogeneous mature forms differing in average Mr from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [35S]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent molecular weight of the molecules or the differences between the various cell lines. beta-D-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.     

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.     

Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo.

The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function.     

Photoaffinity labeling of pituitary gonadotropin-releasing hormone receptors in goldfish (Carassius auratus)

Receptors for GnRH were labeled by use of an iodinated (125I) photoreactive GnRH derivative [D-Lys6-azidobenzoyl]-GnRH. This derivative was found to bind to two classes of GnRH binding sites: high-affinity/low-capacity sites and low-affinity/high-capacity sites. The binding affinity of [D-Lys6-azidobenzoyl]-GnRH was found to be greater than that of D-Lys6-GnRH, but lower than a superactive fish GnRH agonist [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH (sGnRH-A). Analysis of the photoaffinity-labeled goldfish pituitary GnRH receptors by SDS-PAGE and autoradiography indicated the presence of three labeled proteins displaceable by unlabeled sGnRH-A. The first and the most prominently labeled band was a 71,000-Mr protein, the second a 51,000-Mr protein, and the third a minor band of 130,000 Mr. Displacement characteristics of the 71,000- and 130,000-Mr bands were consistent with those of the low-affinity binding sites; displacement of the iodinated ligand from these proteins was achieved only in the presence of 10(-6) M sGnRH-A. The 51,000-Mr band had characteristics similar to those of the high-affinity site; displacement of the labeled ligand was achieved in the presence of 10(-9) M sGnRH-A. These findings provide for the first time some biochemical characterizations of pituitary GnRH receptors in a nonmammalian vertebrate.     

Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila.

A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila. This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B. The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits. Electrophoresis, immunoblot, and [35S]methionine incorporation studies are reported which support the conclusion that the H3 antigen is an acidic protein with an Mr of approximately 52,000 daltons. An induced synthesis of the H3 immobilization antigen was detected within 30 min after a shift from 40 to 28 degrees C. This protein appeared to be synthesized in the microsomal fraction and transferred without cleavage to the cell surface, where it was inserted first into nonciliated regions.     

Extracellular proteins secreted by the basidiomycete Schizophyllum commune in response to carbon source

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.     

Biosynthesis of mammalian DNA ligase

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.     

Macrophage recognition of immune complexes: development and application of novel cell surface labeling procedures

A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex has been prepared for cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas [Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]. The conjugate is a heterodimer of Mr230 000 with linkages to either or both of the heavy and light chains of the antibody, as judged by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. Electron microscopy demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and electron microscopy. Immune complexes were masked with "cold" iodine by use of the endogenous LPO activity. The complexes bound to cells at 4 degrees C as shown by electron microscopy and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling has also been conducted under conditions of substrate deficit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.     

Characterization of the cell surface heterodimer VLA-4 and related peptides

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.     

Tissue distribution, structural characterization, and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity

Mac-3 is a mouse macrophage differentiation antigen defined by a rat anti-mouse monoclonal antibody (MAb),M3/84. The structure, biosynthesis, quantitative surface expression, and distribution of Mac-3 have been studied by radiolabeling and isolation with MAb-Sepharose, saturation binding, absorption, and immunofluorescence flow cytometry. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mac-3 migrates as a diffuse band with average Mr = 110,000. Labeling of intact cells with 125I and accessibility to MAb show it is present at least in part on the cell surface. Saturation labeling with 125I-MAb shows 4.2 X 10(4) cell surface sites on thioglycollate medium-elicited peritoneal macrophages. [35S]Methionine and [3H]glucosamine incorporation into Mac-3 by purified macrophages show it is a glycoprotein synthesized by these cells. Absorption shows Mac-3 is strongest in macrophages, present in lower quantities in lung, liver, bone marrow, and spleen, and undetectable in thymus, lymph node, brain, and heart. Immunofluorescent flow cytometry shows surface expression on thioglycollate-elicited macrophages but not bone marrow, spleen, lymph node, or thymus cell suspensions. Similar amounts of Mac-3 are immunoprecipitated from resident macrophages or macrophages elicited by sterile inflammatory agents, intracellular parasites, or immunomodulators, but the average Mr of Mac-3 varies from 92,000 to 110,000. Mac-3 is synthesized from precursor(s) of Mr = 74,000 and 79,000, identical in the different macrophages. Processing into the mature molecule, which migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a more diffuse band and varies in Mr among macrophage elicited by different agents and to a lesser degree between different preparations of the same type of macrophage, occurs in 15 to 30 min.     

Broadly neutralizing anti-HBV antibody binds to non-epitope regions of preS1

Broadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Biosynthetic precursors and in vitro translation products of the glucose transporter of human hepatocarcinoma cells, human fibroblasts, and murine preadipocytes

Antisera to the human erythrocyte Glc transporter immunoblotted a polypeptide of Mr 55,000 in membranes from human hepatocarcinoma cells, Hep G2, human fibroblasts, W138, and murine preadipocytes, 3T3-L1. This antisera immunoprecipitated the erythrocyte protein which had been photoaffinity labeled with [3H]cytochalasin B, immunoblotted its tryptic fragment of Mr 19,000, and immunoblotted the deglycosylated protein as a doublet of Mr 46,000 and 38,000. This doublet reduced to a single polypeptide of Mr 38,000 after boiling. When Hep G2, W138, and 3T3-L1 cells were metabolically labeled with L-[35S]methionine for 6 h, a broad band of Mr 55,000 was immunoprecipitated from membrane extracts. In pulse-chase experiments, two bands of Mr 49,000 and 42,000 were identified as putative precursors of the mature transporter. The t1/2 for mature Glc transporter was 90 min for Hep G2 cells that had been starved for methionine (2 h) and pulsed for 15 min with L-[35S]methionine. Polypeptides of Mr 46,000 and 38,000 were immunoprecipitated from Hep G2 cells that had been metabolically labeled with L-[35S]methionine in the presence of tunicamycin. This doublet reduced to the single polypeptide of Mr 38,000 after boiling. In the absence of tunicamycin, but not in its presence, mature polypeptide of Mr 55,000 was immunoprecipitated from Hep G2 cells metabolically labeled with D-[3H]GlcN. A polypeptide of Mr 38,000 was observed in boiled immune complexes from the in vitro translation products of Hep G2, W138, and 3T3-L1 cell RNA. Dog pancreatic microsomes cotranslationally, but not posttranslationally, converted this to a polypeptide of Mr 35,000. A model for Glc transporter biogenesis is proposed in which the primary translation product of Mr 38,000 is converted by glycosylations to a polypeptide of Mr 42,000. The latter is then processed via heterogeneous complex N-linked glycosylations to form the mature Glc transporter, Mr 55,000.     

Epidermal growth factor-induced truncation of the epidermal growth factor receptor

NIH-3T3 cells expressing the human epidermal growth factor (EGF) receptor were used in experiments to determine the fate of the EGF receptor in cells continuously exposed to EGF. EGF receptor was immunoprecipitated from cells labeled for 12 h with [35S] methionine in the absence or presence of 10 nM EGF. As expected, a single Mr = 170,000 polypeptide representing the mature EGF receptor was immune-precipitated from control cells. Surprisingly, immune precipitates from EGF-treated cells contained a prominent Mr = 125,000 receptor species, in addition to the Mr = 170,000 mature receptor. The Mr = 125,000 species was shown to be derived from the Mr = 170,000 form by pulse-chase experiments, in which the Mr = 170,000 receptor chased into the Mr = 125,000 form when EGF was included during the chase and by partial proteolysis. Both proteins became extensively phosphorylated on tyrosine residues in immune precipitate kinase assays. Treatment of immune precipitates with endoglycosidase F changed the apparent molecular weight of the Mr = 170,000 receptor to Mr = 130,000 and of the Mr = 125,000 form to Mr = 105,000, indicating that the appearance of the Mr = 125,000 protein was probably due to proteolysis. Antibody against the carboxyl terminus of the mature EGF receptor recognized the Mr = 125,000 protein, whereas antibody against the amino terminus did not. Incubation of cells with leupeptin prior to and during EGF addition inhibited processing to the Mr = 125,000 species. Methylamine and low temperature also inhibited the EGF-induced processing to the Mr = 125,000 form. These data suggest a possible role for proteolysis of the EGF receptor in receptor function.     

Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily.

gp49 is a Mr 49,000 glycoprotein expressed on the surface of mouse bone marrow-derived mast cells, which are progenitors for the major in vivo mast cell subclasses, typified by intestinal mucosal mast cells and serosal mast cells. The amino-terminal amino acid sequence of gp49 was determined after isolation of the solubilized membrane protein by affinity chromatography with the B23.1 anti-gp49 monoclonal antibody. Redundant oligonucleotides were used to isolate a full-length 1.3-kilobase cDNA from a mouse mast cell library. The predicted amino acid sequence contains a signal peptide of 23 residues, an extracellular domain of 215 residues with three potential sites of N-linked glycosylation, a transmembrane domain of 23 residues, and a cytoplasmic tail of 42 residues. Hybridization of the gp49 cDNA was limited to mRNA extracted from those cell types that also bound the B23.1 monoclonal antibody as assessed by cytofluorographic analyses. The predicted extracellular domain of gp49 contains two regions of 48 and 51 amino acids, each flanked by cysteine residues. Both regions meet criteria for being C2-type domains of the immunoglobulin superfamily based upon the alignment of consensus amino acids and their predicted secondary structure organization. Thus, gp49, a membrane glycoprotein preferentially expressed by the progenitor mast cell population, is a new member of the immunoglobulin superfamily.