Identification of integrin-like matrix receptors with affinity for interstitial collagens

Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors.     

Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.     

Comparison of fibronectin receptors from rat hepatocytes and fibroblasts

A cell-surface fibronectin receptor was isolated from primary rat hepatocytes by affinity chromatography on Sepharose conjugated with the cell-binding domain (105 kDa) of fibronectin. The receptor remained bound to the affinity column in the presence of 1 M NaCl but was eluted by 1.5 mM of glycl-arginyl-glycyl-aspartyl-seryl-cysteine peptide or by lowering the pH to 4. The eluted material migrated under nonreducing conditions in sodium dodecyl sulfate-polyacrylamide electrophoresis as two bands: the alpha- and beta-components had apparent Mrs of 155,000 and 115,000, respectively. After reduction the 155-kDa component gave rise to two peptides of Mrs 145,000 and 20,000, while the 115-kDa component shifted migration to an Mr of 130,000. Antibodies specifically recognizing the 155- and 115-kDa proteins from hepatocytes inhibited the attachment of these cells to fibronectin-coated dishes, whereas attachment to dishes coated with collagen or laminin was unaffected. A fibronectin receptor isolated from rat fibroblasts showed closely similar, but not identical, migration in sodium dodecyl sulfate-electrophoresis as the hepatocyte receptor. Furthermore, only the beta-subunit of the fibroblast receptor reacted with the antibodies. The results suggest that distinct alpha-subunits of the fibronectin receptors may be the basis for the different fibronectin-binding properties of these cells.     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Alkaline phosphatase from adult rat femur

Four alkaline phosphatase forms from adult rat femur were distinguished on polyacrylamide gel electrophoresis: two soluble forms of Mr 165,000 and 110,000 in the water extract, and three membrane-bound forms of Mr 130,000, 110,000 and 100,000 extractable with deoxycholate. Alkaline phosphatase after SDS-treatment disintegrated into three kinds of monomers: of Mr 80,000, 65,000 and 50,000. The soluble fraction (extract I) contained subunits of Mr 80,000 and 55,000--whereas the pellet fraction (extract II), subunits of Mr 65,000 and 50,000. Since for native forms only three types of subunits were found it seems that, apart from homodimers, there are also some heterodimers composed of the Mr 65,000 and 50,000 subunits forming the native enzyme of Mr 110,000-115,000. Two denatured monomers: of Mr 80,000 and 50,000 may form two native homodimeric forms of Mr 165,000 and 100,000 while in the pellet two monomers: of Mr 65,000 and 50,000 may correspond to three native alkaline phosphatase forms: of Mr 130,000, 110,000-115,000 and 100,000. Probably the Mr 110,000-115,000 form is a heterodimer composed of subunits of Mr 65,000 and 50,000.     

Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.

Desmosomal proteins (dp1-4) and glycoproteins (dg1-3) have been localised within desmosomes of bovine nasal epithelium by immunogold labelling of ultrathin frozen sections. Beginning in the extracellular space and proceeding through the plaque to the tonofilaments, the following localisations were found. Labelling for the 130,000 and 115,000 Mr glycoproteins (dg2 and dg3) was predominantly in the extracellular space, a location consistent with their proposed adhesive function. The glycoproteins of 175,000-164,000 Mr (dg1) were also found in the extracellular space and in addition had cytoplasmic domains extending throughout the cytoplasmic plaque. The 83,000 Mr protein (dp3) was located along the cytoplasmic face of the membrane and extended into the plaque, whereas an antibody which recognises both the 83,000 Mr protein (dp3) and the 75,000 Mr protein (dp4) gave labelling both in and beyond the plaque. Labelling for the high mol. wt proteins of Mr 250,000 and 215,000 (dp1 and dp2) was largely excluded from the plaque, and was located distally, adjacent to the tonofilaments. Hemidesmosomes could not be labelled with antibodies to dg1-3 or dp3 and 4, but some labelling was obtained with antibody to dp1 and 2.     

Heymann antigen GP330 demonstrates affinity for fibronectin, laminin, and type I collagen and mediates rat proximal tubule epithelial cell adherence to such matrices in vitro.

We have utilized monoclonal antibodies directed against glycoproteins on the surface of proximal tubule epithelial cells (PTEC) to study their interaction with matrix components. PTEC exposed to monoclonal antibodies directed against a 330-kDa cell surface glycoprotein exhibited a significant epitope-specific inhibition of attachment and proliferation on type I collagen-, fibronectin-, laminin-, and gelatin-coated tissue culture surfaces. This effect was not due to antibody toxicity since such cells did not exhibit metabolic dysfunction in suspension cultures and the inhibition could be reversed upon removal of the antibody from the cell surface. Furthermore, detergent-solubilized gp330 demonstrated specific affinity for fibronectin, laminin, and type I collagen which was not inhibited by Arg-Gly-Asp-containing peptides. A monoclonal antibody directed against the receptor epitope was capable of promoting PTEC adherence and growth when such an antibody was immobilized on cell culture dishes. Although gp330 acted as a receptor for matrix proteins in primary cultures of freshly isolated PTEC, this effect was not demonstrable in established cultures. These results suggest that freshly isolated PTEC depend on gp330 for their attachment to matrix molecules while in vitro-adapted PTEC rely on other receptors activated by culture conditions. The affinity of gp330 for matrix molecules may be of pathogenic relevance in the persistence of gp330-containing immune complexes formed in the glomerular capillary wall in experimental membranous nephropathy (Heymann nephritis).     

Aspects of the metabolism of the epidermal growth factor receptor in A431 human epidermoid carcinoma cells.

The biosynthesis and posttranslational metabolism of the epidermal growth factor (EGF) receptor were examined in the A431 human epidermoid carcinoma cell line. Polyclonal antibody against the receptor specifically immunoprecipitated two [35S]methionine-labeled proteins of Mr = 160,000 and 170,000. Pulse chase experiments showed the Mr = 160,000 protein to be a precursor of the Mr = 170,000 protein. Preincubation with tunicamycin resulted in immunoprecipitation of a single band of Mr = 130,000, whereas monensin inhibited maturation to the Mr = 170,000 form. Digestion of the Mr = 160,000 and 170,000 proteins with endoglycosidase H resulted in the appearance of Mr = 130,000 and 165,000 proteins, respectively. Prolonged pulse-chase experiments indicated that the half-life of the receptor is ca. 20 h in the absence of EGF and 5 h in the presence of EGF. Approximately three- to five-fold more phosphate is incorporated into the mature receptor upon addition of EGF, due primarily to increases in levels of phosphotyrosine and phosphoserine. Phosphate was also present on the Mr = 160,000 protein and the Mr = 130,000 protein found in the presence of tunicamycin.     

Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro.

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 x 10(3) Mr and other components. To characterize the 120 x 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 x 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.     

The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies

The receptor for human interferon-gamma (IFN-gamma) was purified from foreskin fibroblasts. Triton X-100 extracts obtained from either intact cells or membrane preparations were passed through an immobilized interferon-gamma column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of eluted fractions revealed a major band of Mr = 95,000 and minor bands of Mr = 80,000 and 60,000. Further purification was obtained by steric exclusion and by lectin chromatography. The purified receptor retained the ability to bind 125I-IFN-gamma with a Kd of 2.2 X 10(-10) M, a value close to that obtained with intact fibroblasts (5 X 10(-10) M). A complex of Mr = 105,000-125,000 was visualized by immunoprecipitation of 125I-IFN-gamma cross-linked to the purified receptor followed by SDS-PAGE and autoradiography. A similar complex was obtained when 125I-IFN-gamma was cross-linked to intact cells. Immunization of mice with the excised SDS-PAGE band of Mr = 95,000 elicited antibodies that blocked the antiviral activity of IFN-gamma and immunoprecipitated the cross-linked complex of 125I-IFN-gamma and its receptor.     

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.     

Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri)

Summary The influence of trout serum on the attachment and spreading of isolated trout hepatocytes maintained in primary culture at different temperatures was evaluated. Hepatocytes were obtained from young rainbow trout (Salmo gairdneri) by collagenase dissociation and maintained in modified Leibowitz L15 medium at 10° or 27° C for 24 h in plastic dishes previously coated with type I bovine collagen. In the absence of serum, fewer than 10% of hepatocytes attached and none of them spread on the collagen substrate. Trout serum at concentrations as low as 1.25% in the medium resulted in a pronounced concentration-dependent increase in hepatocyte attachment, as determined by direct counts by phase contrast microscopy, or by percentage of lactate dehydrogenase activity attached to the dishes after washing away unattached cells. Attachment rates were greater at the lower temperature (10° C). Trout serum also substantially increased the proportion of attached hepatocytes that spread as monolayers on the collagen substrate, especially at 10° C. By comparison, fetal bovine serum had little influence on the attachment or spreading of trout hepatocytes. These studies demonstrate a simple inexpensive method for preparing attached monolayer trout hepatocyte cultures. This procedure may be useful in toxicologic or functional studies in which fish hepatocyte attachment is an operational requirement.     

Glycosylation of the epidermal growth factor receptor and its relationship to membrane transport and ligand binding

Epidermal growth factor (EGF) receptor biosynthesis was examined in an oral squamous cell carcinoma line, NA, which overproduces the receptor to an even greater extent than the widely studied A431 cells. The EGF receptor of NA cells synthesized in the presence of tunicamycin had an apparent molecular weight of 130,000. The nascent protein in untreated cells was cotranslationally glycosylated to Mr 160,000 and further processed to Mr 170,000. The endo-beta-N-acetylglucosaminidase H (Endo H) digestion analysis revealed the presence of high mannose type oligosaccharide on the Mr 170,000 mature receptor. We extended the analysis by correlating the biosynthesis with the acquisition of binding activity. The unglycosylated Mr 130,000 receptor and the Mr 160,000 receptor seen after pulse-labeling had no EGF binding activity, whereas the Mr 160,000 receptor seen after chase-incubation and the Mr 170,000 receptor had binding activity. Thus, not only glycosylation but also some oligosaccharide processing is apparently necessary for the EGF binding. Treatment with processing inhibitors, such as monensin, swainsonine and 1-deoxynojirimycin, affected neither receptor transport to the plasma membrane nor binding activity. Inhibition by 1-deoxynojirimycin is thought to be incomplete since the surface receptor in treated cells had the same molecular weight as that in control cells. An Mr 160,000 receptor without binding activity accumulated in the intracellular fraction in the presence of brefeldin A, an inhibitor of intracellular transport. Thus, the EGF binding activity is thought to be acquired after the brefeldin A-sensitive process but prior to the swainsonine-sensitive mannose removal in NA cells.     

Characterization of soluble forms of NCAM

Neural cell adhesion molecule (NCAM) has been described as a family of membrane glycoproteins. However, soluble NCAM immunoreactivity has long been recognized. We here show that soluble NCAM is composed of two quantitatively major polypeptides of Mr 180,000 and 115,000 and two minor components of Mr 160,000 and 145,000. Soluble NCAM was immunochemically identical to membrane NCAM, was polysialylated and carried the HNK-1 epitope. It only constituted 0.8% of total NCAM in newborn rat brain. Soluble NCAM appeared in neuronal cell culture medium 15-30 min after the start of synthesis preceding accumulation of membrane-associated NCAM on the cell surface. This indicates that soluble NCAM contains a secreted component.     

Adsorption from fetal calf serum of collagen-like proteins which bind fibronectin and promote cell attachment

Baby hamster kidney cells were seeded onto Western blots of fetal serum proteins which had been extracted from several foreign surfaces. This revealed that the major cell adhesive proteins adsorbed onto these surfaces from fetal serum were (1) fibronectin of Mr 220,000 Da and (2) vitronectin of Mr 65,000 and 78,000 Da. Two minor bands of cell attachment were observed at Mr 153,000 and Mr 134,000 Da in the fetal serum proteins extracted from heparin-agarose and serotonin-agarose. However, by exposing the Western blots of separated proteins to a second round of serum proteins, prior to cell blotting, very strong cell adhesive bands were revealed at Mr 153,000, 134,000, and 120,000 Da. By (i) modifying the composition of the serum proteins used to treat the Western blots, (ii) using specific antibodies to fibronectin, and (iii) using radiolabeled fibronectin, it was conclusively demonstrated that the new cell adhesive bands owed their increased cell attachment activity to secondary binding of fibronectin. The new bands were shown (i) to be trypsin sensitive and collagenase sensitive and therefore to be collagen-like proteins and (ii) to react negatively in immunoblots using anti-fibronectin, anti-vitronectin, anti-fibrinogen, anti-fetuin or anti-thrombospondin. In SDS-PAGE (i) the Mr 120,000-Da protein comigrated with the alpha 2-chain of Type I collagen, (ii) the Mr 134,000-Da protein comigrated with the alpha 1-chain of Type I collagen, and (iii) the Mr 153,000-Da protein comigrated with the pN-alpha 1-chain of Type III collagen. Since the novel collagen-like proteins acted as strong sites of cell attachment on nitrocellulose blots by binding fibronectin, they might well promote cell attachment on the foreign surfaces from which they were extracted.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Switching from differentiation to growth in hepatocytes: control by extracellular matrix.

Studies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non-adhesive plastic dishes that were pre-coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell-cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver-specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down-regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell-cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.     

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.     

The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture

Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents.