Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is approximately 80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.     

Nectin-like molecule 1 is a glycoprotein with a single N-glycosylation site at N290KS which influences its adhesion activity

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3, from now on referred to as NECL1, is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule which has Ca(2+)-independent homo- or heterophilic cell-cell adhesion activity and plays an important role in the formation of synapses, axon bundles and myelinated axons. Here we first detected the expression of NECL1 in human fetal and adult brains, and mouse brains at different developmental stages. The results indicated that two bands with molecular weights of about 62 kDa and 48 kDa were found in human fetal brain, while only one band with a molecular weight of about 48 kDa was found in human adult brain; two bands with molecular weights of about 62 kDa and 48 kDa whose expression level gradually increased were also found from mouse E16 to P14, while only one band with a molecular weight of about 48 kDa was found from P14. Bioinformatics analysis showed there were two putative N-glycosylation sites within human NECL1 at positions N25LS and N290KS and within mouse Necl1 at positions N23LS and N288KS, respectively. There was no O-glycosylation site in either human NECL1 or mouse Necl1. Based on the results of N-Glycosidase F treatment with human fetal brain tissue and lysates from transient transfection with human wild-type or glycosylation site mutant NECL1 in 293ET cells, we demonstrated that human NECL1 is an N-linked glycoprotein with a single glycosylation site at position N290KS. Cell aggregation assay further showed there was an increased adhesion activity after the glycosylation site mutation of NECL1 molecule.     

Properties of the PC-1 molecule

The technique of cell-surface iodination, followed by immuno-precipitation withPC-1.1 antiserum, was applied to normal spleen cells and to MOPC-70A BALB myeloma cells. The results indicate that the cell-surface component bearing PC-1 alloantigen has a molecular weight of from 105,000 to 110,000 daltons and does not resemble any constituent of plasma membranes or MuLV-type virus so far categorized by similar methods. The PC-1 specificity of the molecule was confirmed by comparison of the spleen cells of two mouse strains with spleen cells of their respective PC-congenic partner strains. MOPC-70A myeloma cells, but not spleen cells, yield a fainter band in the PC-1 position in controls in which antiserum is omitted, but peptide maps show that this similarly placed band has no relation to the PC-1 molecule.Abbreviations used in this paper are as follows PC plasma cell - B6 C57BL/6 - SDS/PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - a anti - NMS normal mouse serum - MuLV murine leukemia virus - Ig immunoglobulin     

Identification of an 80 kDa glycoprotein located at sites of close apposition between skeletal muscle cells

An 80 kDa glycoprotein was isolated from adult frog skeletal muscle by concanavalin (Con A) affinity chromatography and electrophoretic separation by molecular mass. Characteristics of the 80 kDa glycoprotein are that it: 1) binds non-covalently to gelatin-agarose or some other protein(s) bound to gelatin-agarose, 2) does not bind wheat germ agglutinin, 3) appears only at 80 kDa in both reducing and non-reducing electrophoretic separations, 4) is present in skeletal muscle but absent in smooth muscle and cardiac muscle, 5) is not collagenase or hyaluronidase-sensitive, and 6) is antigenically similar to a protein in embryonic chicken skeletal muscle. It was used to generate a polyclonal antiserum which was affinity-purified and used for immunolocalization. Indirect immunofluorescence procedures showed the antigen to be present on the surface of the skeletal muscle cells and concentrated at sites where cells are closely apposed to one another. Preparations in which adult muscle cells were depleted of basement membrane and endomysial proteins did not reduce the amount of 80 kDa protein present in skeletal muscle. These data indicate that this is a cell surface glycoprotein that may mediate attachment of the cell to extracellular proteins at sites where adjacent skeletal muscle cells are apposed.     

Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.     

Nectin-like molecule 1 is a glycoprotein with a single N-glycosylation site at N290KS which influences its adhesion activity

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3, from now on referred to as NECL1, is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule which has Ca²⁺-independent homo- or heterophilic cell-cell adhesion activity and plays an important role in the formation of synapses, axon bundles and myelinated axons. Here we first detected the expression of NECL1 in human fetal and adult brains, and mouse brains at different developmental stages. The results indicated that two bands with molecular weights of about 62 kDa and 48 kDa were found in human fetal brain, while only one band with a molecular weight of about 48 kDa was found in human adult brain; two bands with molecular weights of about 62 kDa and 48 kDa whose expression level gradually increased were also found from mouse E16 to P14, while only one band with a molecular weight of about 48 kDa was found from P14. Bioinformatics analysis showed there were two putative N-glycosylation sites within human NECL1 at positions N25LS and N290KS and within mouse Necl1 at positions N23LS and N288KS, respectively. There was no O-glycosylation site in either human NECL1 or mouse Necl1. Based on the results of N-Glycosidase F treatment with human fetal brain tissue and lysates from transient transfection with human wild-type or glycosylation site mutant NECL1 in 293ET cells, we demonstrated that human NECL1 is an N-linked glycoprotein with a single glycosylation site at position N290KS. Cell aggregation assay further showed there was an increased adhesion activity after the glycosylation site mutation of NECL1 molecule.     

Membrane receptors for murine leukemia viruses: characterization using the purified viral envelope glycoprotein, gp71.

The 71,000 dalton glycoprotein (gp71) purified from Rauscher murine leukemia virus (R-MuLV) by affinity chromatography specifically binds to murine but not other mammalian cells in culture. Binding is prevented by specific antiserum raides to gp71 (anti-gp71). The binding assay as described in this report can detect receptors on as few as 300 murine cells, and with 1 X 10(5) cells gives significant binding with 30 sec. The results show that the purified glycoprotein retians biologic activity and can form a stable complex with specific receptors on mouse cell membranes. The assay can therefore be used to characterize the nature of the cellular receptors that are essential for leukemia virus infection. Purified gp71 binding to mouse cells is prevented if the cells are actively producing related ecotropic type C viruses, presumably because the receptors are occupied and are not available to bind exogenously applied gp71. The binding of gp71 to murine cells is enhanced by the presence of calcium ions and low pH. Binding studies performed using an excess of 125I-gp71 indicate the NIH/3T3 cells bind approximately 5.3 X 10(5) molecules of 125I-gp71 per cell.     

The 32-kDa glycoprotein of Chlamydia trachomatis is an acidic protein that may be involved in the attachment process

Abstract The 32-kDa glycoprotein of Chlamydia trachomatis was shown to have a p I of 6.2 to 6.4 which distinguished this protein from the chlamydial histone-like protein of similar molecular mass that has a p I of > 10. The initial interaction of the glycan of 32 kDa glycoprotein and HeLa cells was also investigated. Glycan was cleaved from the protein backbone by N -glycanase and radiolabeled with tritium by sodium borohydride reduction. Competition assays showed the binding of glycan to HeLa cells was inhibited by galactose, mannose, and N -acetylglucosamine but not by sedoheptulose and fructose. Untreated and UV-treated organisms inhibited the binding, while heat-inactivated organisms did not. Binding was blocked by rabbit antiserum against whole organisms but not by rabbit anti-155-kDa antiserum or monoclonal antibodies against the lipopolysaccharide and major outer membrane protein.     

Preparation and properties of an antiplasma cell serum directed against a defined plasmacytoma cell population.

Heterologous antisera were prepared against a subpopulation of MOPC-104E tumor cells obtained by centrifugation on discontinuous BSA gradients as well as against cells from the whole tumor mass. The gradient-separated cells were more effective than the cells from the whole tumor mass in eliciting antisera not only higher titer, but also with greater specificity for plasmacytoma antigens. The unabsorbed antiserum prepared against the gradient-separated plasmacytoma population was cytotoxic for murine lymphoid cells, but not for murine kidney, liver, or brain cells. After in vitro absorption with murine thymocytes and removal of anti-immunoglobulin activity by affinity chromatography, the antiserum was found to be reactive against plasmacytoma cells, but was no longer cytotoxic for murine thymus or unstimulated spleen cells. This absorbed antiserum was also cytotoxic for LPS-, but not PHA- or Con A-stimulated normal murine spleen cells.     

Type-Common CP-1 Antigen of Herpes Simplex Virus Is Associated with a 59,000-Molecular-Weight Envelope Glycoprotein

The CP-1 antigen of herpes simplex virus type 1 (HSV-1) is a glycoprotein found in the soluble portion of infected cells, in detergent extracts of infected cell membranes, and in the envelope of purified virus. Antisera were prepared against a further purified form of CP-1 prepared from HSV soluble antigen mix; a glycoprotein, gp52, isolated from detergent-treated infected cells; and detergent extracts of purified virus. Each of the antisera reacted with CP-1 to give a single immunoprecipitin band of identity, and each antiserum neutralized the infectivity of HSV-1 and HSV-2. Our results suggested that the type-common determinants involved in the stimulation of neutralizing antibody resided on a 52,000-molecular-weight (52K) glycoprotein. The envelope of HSV contains several glycoproteins: one component at 59K and a complex of two or three components at 130K, none of which corresponds in molecular weight to gp52. Using the antisera as immunological probes, we performed pulse-chase experiments with [(35)S]methionine-labeled HSV-1-infected cells and followed the disposition of the glycoproteins during the infectious cycle. Each antiserum immunoprecipitated a (35)S-labeled 52K protein from lysates of cells pulse-labeled at 5 h after infection. By 10 h, the label was chased into a 59K protein also precipitable by each of the three antisera. The results suggest that gp52 is a precursor of gp59 and that the latter corresponds in molecular weight to one of the major glycoproteins of the virion envelope.     

Neutral alpha-glucosidase from human kidney. Molecular and immunological properties. Relationship with intestinal glucoamylase.

Some molecular properties of the purified neutral alpha-glucosidase from human kidney were studied. The enzyme is a glycoprotein with high molecular weight (315000-352000 according to the method used). Its sedimentation coefficient is 12.9S. It exhibits at least three peaks of activity in isoelectric focusing experiments. This heterogeneity appears to be related to sialic acid residues from the carbohydrate moiety. An anti-human renal alpha-glucosidase antiserum was raised from rabbit. The antiserum effect on human intestinal maltases was studied in immunodiffusion experiments. An identity pattern was observed between renal neutral alpha-glucosidase and intestinal glucoamylase. No precipitation occurred with intestinal sucrase. Renal neutral alpha-glucosidase and intestinal glucoamylase were both completely precipitated by the antiserum, their maltase activity being only slightly inhibited in the antigen-antibody complex. From their molecular and immunological properties a large homology appears between human renal alpha-glucosidase and intestinal glycoamylase.     

Large scale isolation of bovine and pig zonae pellucidae: Chemical,immunological, and receptor properties

A procedure is described for isolating milligram quantities of bovine and porcine zonae pellucidae, uncontaminated by follicle cells or their processes. On SDS-polyacrylamide gel electrophoresis the isolated bovine zona material formed one major glycoprotein band with an estimated molecular weight of approximately 100,000 daltons and two minor lower molecular weight components. The isolated pig zonae formed only one glycoprotein band with a molecular weight of approximately 62,000 daltons. Rabbit antisera raised against the isolated zonae were zona-specific and formed only a single precipitin line against the heat-solubilized zonae on immunoelectrophoresis. An adjuvant was not required for high-antibody titers. High titers were also obtained by injection of the dog and rhesus monkey. Anti-zona antibody was detected by immunofluorescence, zona-coating, double-immunodiffusion, and the inhibition of spermbinding to eggs, including those of human origin. Antigenic and sperm receptor properties were stable at 100°C for five minutes, but some activity was lost after longer exposure. The serum antibody produced by rabbits immunized with pig zonae was predominantly IgG and cross-reacted with the zonae of a variety of other species, including primates. Pregnancy was inhibited in female rabbits immunized with pig zona preparations.     

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.     

Structural and immunological characterization of the amino-terminal domain of mammalian neural cell adhesion molecules

The neural cell adhesion molecules (N-CAMs) are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000 and 140,000 with an additional form at 120,000 in murine brain. In embryonic brain, N-CAMs are represented by a highly sialylated form with an apparent molecular weight greater than 180,000. We have used monoclonal antibodies that cross-react with N-CAMs of various mammalian species to purify N-CAMs from adult murine and bovine brains and from embryonic murine brains. We determined the amino acid sequences of the amino-terminal domains of these molecules: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly-Glu-Ile-Ser-Val-Gly-Glu-Ser. This sequence is highly conserved among all three forms of adult murine brain N-CAM as well as embryonic murine brain N-CAM and adult bovine brain N-CAM. Based on this sequence, we synthesized an undecapeptide and used it to raise a site-directed polyclonal antiserum. This antiserum reacted with the intact N-CAM in liquid phase radioimmunoassays, immunoblotting experiments, and immunofluorescent labeling of cells. The antiserum also reacted with N-CAMs in extracts of brain tissues from different species, confirming the highly conserved nature of the amino-terminal domain of mammalian N-CAMs. Immunofluorescence experiments indicated that this domain resides on the outer surfaces of cells that express N-CAMs, in both primary neuronal cell culture and in cell lines.     

Characterization of a high-molecular-weight protein immunoprecipitated by platelet-derived growth factor antisera

We previously demonstrated that a high-molecular-weight glycoprotein could be immunoprecipitated from metabolically labeled U-2 OS cells with platelet-derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF-like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U-2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. 1) It is a PDGF binding protein that is unrelated to alpha 2-macroglobulin. 2) It is phosphorylated in response to PDGF stimulation. 3) It is immunoprecipitated by phosphotyrosine antibodies. 4) It is not a substrate of epidermal growth factor-induced tyrosine kinase activity. These studies indicate that high-molecular-weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF-induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.     

Detection of a nerve-specific membrane protein on differentiating PCC3/A/1 cells

Attempts were made to prepare monoclonal antibodies specifically reactive with cell surface components of a murine neuroblastoma cell line, Neuro 2a. One of the antibodies (1c2) reacts with a varying proportion of in vitro cultivated Neuro 2a cells, but does not react with murine embryonal carcinoma cell lines (PCC3/A/1 and F9) or with a murine fibroblast line (LM). This antibody selectively stains a subpopulation of nerve cells in murine adult central nervous system, e.g. granular cells in cerebellar cortex. Immunoaffinity purification of adult brain and Neuro 2a plasma membrane fractions with the antibody resulted in an electrophoretically pure protein of approx. 28 kD molecular weight as estimated by SDS-PAGE. Although this antigen is absent from PCC3/A/1 embryonal carcinoma cells, it can be demonstrated after 9 days of growth and differentiation under low density conditions by indirect immunoperoxidase staining. This monoclonal antibody may prove useful in further analysis of neural tissue development.     

Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate-Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose-detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5'-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000-130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.     

Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus.

The attachment of lymphocytic choriomeningitis virus (LCMV) to murine and primate cell lines was quantitated by a fluorescence-activated cell sorter assay in which binding of biotinylated virus was detected with streptavidin-fluorescein isothiocyanate. Cell lines that were readily infected by LCMV (e.g., MC57, Rin, BHK, Vero, and HeLa) bound virus in a dose-dependent manner, whereas no significant binding was observed to lymphocytic cell lines (e.g., RMA and WIL 2) that were not readily infected. Binding was specific and competitively blocked by nonbiotinylated LCMV. It was also blocked by LCMV-specific antiserum and a neutralizing monoclonal antibody to the virus glycoprotein GP-1 but not by antibodies specific for GP-2, indicating that attachment was likely mediated by GP-1. Treatment of cells with any of several proteases abolished LCMV binding, whereas phospholipases including phosphatidylinositol-specific phospholipase C had no effect, indicating that one or more membrane proteins were involved in virus attachment. These proteins were characterized with a virus overlay protein blot assay. Virus bound to protein(s) with a molecular mass of 120 to 140 kDa in membranes from cell lines permissive for LCMV but not from nonpermissive cell lines. Binding was specific, since unlabeled LCMV, but not the unrelated enveloped virus herpes simplex virus type 1, competed with 125I-labeled LCMV for binding to the 120- to 140-kDa band. The proteinaceous nature of the LCMV-binding substance was confirmed by the lack of virus binding to proteinase K-treated membrane components. By contrast, glycosidase treatment of membranes did not abolish virus binding. However, in membranes treated with endoglycosidase F/N-glycosidase F, and/or neuraminidase and in membranes from cells grown in tunicamycin, the molecular mass of the LCMV-binding entity was reduced. Hence, LCMV attachment to rodent fibroblastic cell lines is mediated by a glycoprotein(s) with a molecular mass of 120 to 140 kDa, with complex N-linked sugars that are not involved in virus binding.     

A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity

Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.     

Fetuin: a serum component associated with rat Sertoli and spermatogenic cells in coculture

Cocultures of rat Sertoli-spermatogenic cells plated in a culture medium supplemented with 10% fetal bovine serum for 6-12 h and then maintained in serum free, hormone/growth factor-supplemented medium accumulated an acidic glycoprotein of molecular weight of 68,000 dalton (68 kD) and isoelectric point range of about 4.2-3.5. Anion exchange chromatography has allowed the partial purification of this protein, which consists of a major protein band of 68 kD and two minor, low molecular weight components. A rabbit antiserum raised against the 68 kD component also crossreacts with the two low molecular weight components, thus suggesting that these two minor components are antigenically related to the 68 kD protein. The 68 kD protein has been identified as fetuin, the major component of fetal bovine serum, based on similar molecular weight, isoelectric point, immunoreactivity and trypsin inhibitory activity. Labeling experiments with [14C]amino acid mixture show that 68 kD protein is not synthesized by cocultured rat Sertoli and spermatogenic cells. Immunocytochemistry and Western blot approaches carried out under various experimental conditions support the view that the fetuin-68 kD protein is taken up from serum by both Sertoli cells and pachytene spermatocytes. Because fetuin 1) behaves as a carrier protein for growth factors, 2) has protease inhibitory activity, 3) is preferentially internalized by Sertoli cells and pachytene spermatocytes and 4) fetal bovine serum-supplemented medium impairs spermatogenic cell viability, there is a need to further define appropriate conditions for optimizing long-term viability and differentiation of spermatogenic cells in vitro.