Influenza A virus infection of human middle ear cells in vitro.

HYPOTHESIS: Human-derived normal middle ear mucosal cells can be harvested and cultured and will support influenza A virus (INF A) infection. STUDY DESIGN: Protocols for the collection and in vitro culture of middle ear mucosal cells were developed and used to investigate the effects of INF A infection as it relates to the pathogenesis of otitis media. MATERIALS AND METHODS: Middle ear mucosa was harvested during surgeries that opened the normal middle ear. Middle ear mucosal cells were plated and grown in collagen-coated dishes. Cells were characterized before and after INF A exposure using phase-contrast and immunofluorescence microscopy as well as reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 18 gene expression and INF A. RESULTS: Primary cultures of human middle ear epithelial cells were established. Prolonged growth of middle ear cells yielded a second cell type that failed to stain for cytokeratin on immunofluorescence but continued to produce positive RT-PCR results on cytokeratin 18 analysis. After INF A exposure, cytological changes and immunofluorescence staining showed cellular infection. RT-PCR analysis using INF A-specific primers showed positive results for up to 72 hours after viral exposure. CONCLUSIONS: Primary cultures of human middle ear mucosal cells have been established. Two distinctly different cell culture systems have been developed: 1) middle ear epithelial cells and 2) either dedifferentiated epithelial cells or fibroblasts. Exposure of both cell types to INF A demonstrates that each can support cellular infection and viral replication. These models should be useful for studies of the pathogenesis of virus-mediated otitis media.

An intramembrane modulator of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling.

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

Post-transcriptional regulation of a milk membrane protein, the sialomucin complex (Ascites sialoglycoprotein (ASGP)-1/ASGP-2, rat muc4), by transforming growth factor beta.

Sialomucin complex (SMC, Rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, which can act as a ligand for the receptor tyrosine kinase ErbB2. SMC is highly expressed on the surface of ascites 13762 rat mammary adenocarcinoma cells, approximately 100 times the level in lactating mammary gland and 10(4) times that in virgin mammary gland. SMC is sharply increased at mid-pregnancy in a manner similar to beta-casein. Unlike beta-casein, SMC appears to be regulated post-transcriptionally. Its transcript is present in both virgin and pregnant mammary tissue, and SMC synthesis is induced rapidly in cultured primary mammary epithelial cells from either normal pregnant or virgin rats. SMC protein, but not transcript, levels are significantly reduced when mammary cells are cultured in Matrigel, a reconstituted basement membrane which stimulates casein expression. SMC precursor is synthesized in Matrigel at a 10-fold lower rate. Matrigel has no effect on either the level of SMC or its transcript in cultured 13762 mammary tumor cells. The Matrigel effect on primary mammary and 13762 cells is mimicked by transforming growth factor beta, a component associated with this complex matrix. These results indicate that SMC is a novel product of normal mammary gland and milk, which is post-transcriptionally regulated by transforming growth factor beta in normal mammary gland, but not in 13762 mammary adenocarcinoma cells.

The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products.

Malignant transformation is associated with changes in the glycosylation of cell surface proteins. For example, the N-linked oligosaccharides containing the [GlcNAc beta(1,6)Man] branch are increased after transformation of many cell types by a number of tumor viruses and oncogenes which induce the expression of N-acetylglucosaminyl-transferase V (GlcNAc-T V), the enzyme that adds this branch. A large percentage of human breast carcinomas have increased N-linked beta(1,6) branches on glycoproteins, while up to 30% of breast carcinomas have amplified the oncogene her-2/neu (erb-B2). We tested the hypothesis that expression of her-2/neu stimulates GlcNAc-T V gene expression and increases the beta(1,6) branching of N-linked oligosaccharides. We found that neu-transformed NIH3T3 cells have a threefold increase in GlcNAc-T V enzyme activity and increased beta(1,6) branching on a specific set of glycoproteins. Promoter/reporter experiments showed that her-2/neu stimulates transcription from the human GlcNAc-T V promoter and that the her-2/neu response element was located about 400 bp 5' of the transcription initiation site and includes three Ets transcription factor binding sequences. Co-transfections with dominant-negative Raf and Ets expression plasmids demonstrated that the transcriptional activation of the GlcNAc-T V promoter by neu is mediated by the Ras-Raf-Ets signal transduction pathway.

Characterization of the expression and steroid hormone control of sialomucin complex in the rat uterus: implications for uterine receptivity.

The sialomucin complex (SMC), originally isolated as a heterodimeric glycoprotein complex from membranes of ascites sublines of a highly metastatic mammary adenocarcinoma, consists of a high Mr mucin subunit (ASGP-1, ascites sialoglycoprotein) and a transmembrane subunit (ASGP-2) with two epidermal growth factor-like domains. SMC has been characterized in the mammary gland, where it is present in both membrane and nonmembrane (soluble) forms, the latter lacking its transmembrane domain. SMC in the mammary gland is observed during pregnancy and lactation, but not in the virgin gland, and is regulated by a posttranscriptional mechanism. Both membrane and nonmembrane forms of SMC are found in rat uterus, also as a complex of ASGP-1 and ASGP-2. Immunocytochemical analyses indicate that the primary site of expression is at the luminal surface of the endometrium. Approximately 40% of the SMC, corresponding to the nonmembrane fraction, is removed by rinsing uterine preparations with saline, indicating that the soluble form is adsorbed loosely to the cell surface. In contrast to mammary gland, SMC is most highly expressed in the virgin animal, and its expression varies during the estrous cycle with the steady state level of transcript. The complex is present in a location consistent with steric inhibition of blastocyst implantation and is abruptly lost at the beginning of the period of receptivity for implantation. Expression of SMC in the uterus is regulated by estrogen and progesterone and is inversely correlated with receptivity. Both implantation and loss of SMC expression can be blocked with RU486. We propose that the down-regulation of SMC and its loss from the apical surface of the rat uterine lining contribute to the generation of the receptive state for uterine implantation. Furthermore, the presence of both membrane and soluble SMC at the luminal surface of the endometrium may provide a model for its proposed protective function in other accessible and vulnerable epithelia.

Sialomucin complex in the rat respiratory tract: a model for its role in epithelial protection.

The pulmonary epithelium has a multitude of specialized functions, which depend on regulated growth and differentiation of several cell types. One such function is the synthesis and secretion of mucins, which offer the epithelium protection from and a means for removal of noxious environmental factors. Sialomucin complex (SMC) is a heterodimeric glycoprotein consisting of a mucin subunit (ASGP-1, ascites sialoglycoprotein-1) and a transmembrane protein (ASGP-2) with two epidermal-growth-factor-like domains. SMC was originally discovered in a highly metastatic rat mammary adenocarcinoma and has been implicated in metastasis and in the protection of the tumour cells from natural killer cells. It can also act as a ligand for the receptor tyrosine kinase 185(neu), suggesting that it is bifunctional as well as heterodimeric. SMC is expressed on the epithelium of rat conducting airways, with the highest levels occurring in the proximal trachea and progressively decreasing into the bronchioles. Airway SMC consists of two forms: a soluble form that lacks the C-terminal cytoplasmic and transmembrane domains and accounts for about 70% of the total, and a membrane-associated form that has the C-terminal domains. Immunocytochemical analyses show that SMC is predominantly present on the apical surfaces of the airway epithelium, but not in goblet cells. Soluble form can be removed from the trachea by rinsing, suggesting that a fraction of the protein is adsorbed to the apical surface. Based on these results, we propose a protective mechanism in which membrane and soluble forms of SMC are produced by airway luminal epithelial cells to provide a cell-associated epithelial glycoprotein barrier that also serves as an interface with flowing mucus. In support of this mechanism, we demonstrated secretion of soluble SMC by primary cultures of tracheal epithelial cells. This model suggests that SMC is a critical element in the protective barrier of the airway epithelium.

Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene.

Transformation of baby hamster kidney fibroblasts by the Rous sarcoma virus causes a significant increase in the GlcNAcbeta(1, 6)Man-branched oligosaccharides by elevating the activity and mRNA transcript levels encoding N-acetylglucosaminyltransferase V (GlcNAc-T V). Elevated activity and mRNA levels could be inhibited by blocking cell proliferation with herbimycin A, demonstrating that Src kinase activity can regulate GlcNAc-T V expression. 5' RACE analysis was used to identify a 3-kilobase 5'-untranslated region from GlcNAc-T V mRNA and locate a transcriptional start site in a 25-kilobase pair GlcNAc-T V human genomic clone. A 6-kilobase pair fragment of the 5' region of the gene contained AP-1 and PEA3/Ets binding elements and, when co-transfected with a src expression plasmid into HepG2 cells, conferred src-stimulated transcriptional enhancement upon a luciferase reporter gene. This stimulation by src could be antagonized by co-transfection with a dominant-negative mutant of the Raf kinase, suggesting the involvement of Ets transcription factors in the regulation of GlcNAc-T V gene expression. The src-responsive element was localized by 5' deletion analysis to a 250-base pair region containing two overlapping Ets sites. src stimulation of transcription from this region was inhibited by co-transfection with a dominant-negative mutant of Ets-2, demonstrating that the effects of the src kinase on GlcNAc-T V expression are dependent on Ets.

Regulation of N-acetylglucosaminyltransferase V and Asn-linked oligosaccharide beta(1,6) branching by a growth factor signaling pathway and effects on cell adhesion and metastatic potential.

Recent evidence demonstrates that the changes in the size of N-linked oligosaccharides that correlate with cell transformation and tumorigenicity are due at least in part to the regulation of expression of a glycosyltransferase involved in the branching of N-linked structures, N-acetylglucosaminyltransferase V or GlcNAc-T V. Studies have shown that the increases in GlcNAc-T V expression after oncogenic transformation are most likely caused by direct effects on the GlcNAc-T V promoter by the Ets family of transcriptional activators, which are up-regulated by a cellular proliferation signaling pathway. This pathway begins with growth factor receptors that activate tyrosine kinases at the cell surface and proceeds through src, ras, and raf. Additional evidence for the association between cellular proliferation and GlcNAc-T V expression will be presented, as well as a discussion of the effects of beta(1,6) branching on several of the phenotypes of oncogenically transformed cells, including metastatic potential.

Reversible disruption of cell-matrix and cell-cell interactions by overexpression of sialomucin complex.

Sialomucin complex (SMC) is a large, heterodimeric glycoprotein complex composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits and expressed abundantly on the cell surface of ascites 13762 rat mammary adenocarcinoma cells. We have isolated recombinant cDNAs containing different numbers of ASGP-1 mucin repeats, which can be expressed as protein products with variable lengths. To study the anti-adhesive effect of SMC, these cDNAs were transfected into human cancer cell lines. Using a tetracycline-responsive, inducible expression system, we demonstrated that the overexpression of SMC induces morphology changes, cell detachment, and cell-cell dissociation of transfected A375 human melanoma cells in culture. The transition between the adherent and suspension states of the cells is fully reversible and dependent on the SMC expression level. The anti-adhesion effect of SMC was further analyzed kinetically by measuring the cell adhesion of transfected A375 melanoma and MCF-7 breast cancer cell lines to fibronectin, laminin, and collagen IV, demonstrating that SMC disrupts integrin-mediated cell adhesion to extracellular matrix proteins. The degree of this anti-adhesion effect was dependent on the number of mucin repeats in the SMC molecule as well as the level of cell surface expression.

Sialomucin complex, a heterodimeric glycoprotein complex. Expression as a soluble, secretable form in lactating mammary gland and colon.

Ascites 13762 rat mammary adenocarcinoma cells express abundantly on their cell surfaces a heterodimeric glycoprotein complex composed of a sialomucin ascites sialoglycoprotein (ASGP)-1 and a transmembrane subunit ASGP-2. The latter, which contains two epidermal growth factor-like domains, binds the receptor tyrosine kinase p185(neu), suggesting that the complex is bifunctional as well as heterodimeric. Immunoblot analyses using monoclonal antibodies prepared against the complex demonstrate high levels of expression in rat lactating mammary gland and colon. Immunolocalization studies with anti-ASGP-2 indicate that ASGP-2 is present in these two tissues in the apical regions of secretory epithelial cells. Both mammary gland and colon contain a soluble, secretable form of ASGP-2, which is not found in the ascites cells; milk and mammary gland also have the membrane form. Immunoblot analyses using a COOH-terminal-specific polyclonal antibody indicate that the soluble form of ASGP-2 is missing its COOH-terminal domains. Both the soluble and membrane forms of ASGP-2 are similar to the membrane-associated form from the 13762 adenocarcinoma with respect to Mr, antigenicity, and association with ASGP-1. The presence of ASGP-1 in milk suggests that it is a candidate for the uncharacterized high Mr milk mucin, MUCX. ASGP-2 expression is up-regulated in mammary gland during pregnancy, because it is undetectable in virgin and early pregnant rats but abundant in the gland from late pregnant and lactating animals. However, compared with the lactating mammary gland, the 13762 ascites cells overexpress ASGP-2 by more than 100-fold, which may contribute to their malignancy. These combined results indicate that sialomucin complex is a unique secreted product in the mammary gland and colon, whose behavior is different from that in the mammary ascites tumors, and which may play important roles in mammary and intestinal physiology.

GlcNAc-transferase V and core 2 GlcNAc-transferase expression in the developing mouse embryo.

UDP-GlcNAc:Manalpha1-6Manbeta-R beta1-6-N-acetylglucosaminyltransferase V (GlcNAc-TV) and UDP-GlcNAc:Galbeta1-3GalNAc-R beta1-6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) are Golgi enzymes that catalyse the biosynthesis of beta1-6GlcNAc-branched intermediates in the N- and O-linked biosynthesis pathways, respectively. The activities of these enzymes change during haematopoiesis, embryo-carcinoma cell differentiation and following malignant transformation, but little is known about their expression in normal adult tissues and during embryogenesis. We have examined the expression of GlcNAc-TV and core 2 GlcNAc-T in sections of post-implantation mouse embryos by in situ RNA hybridization. The two enzymes showed distinct temporal and spatial patterns of expression. Core 2 GlcNAc-T mRNA was widely expressed at embryonic day (E) 7, and became restricted to a subset of mucin- and cartilage-producing tissues at E11.5 through to E17.5. GlcNAc-TV transcripts were absent at E7, became expressed throughout E9.5 embryos, and then progressively restricted to regions of the developing central nervous system and to specialized epithelia of skin, intestine, kidney, endocrine tissues and respiratory tract. In the adult gonads, GlcNAc-TV transcripts were excluded from germ cells, but were detected in the follicular and testicular cells. Leukoagglutinin (L-PHA)-reactive oligosaccharides co-localized with GlcNAc-TV transcripts in skin, kidney and intestine, but brain showed unexpectedly low overall staining punctuated by bright staining of the vascular endothelium. A common feature of cells in basal epithelia and in the cortical neural epithelium is the capacity to migrate, a cellular function which may require GlcNAc-TV-dependent glycoconjugates.

Expression of N-acetylglucosaminyltransferase V mRNA in mammalian tissues and cell lines.

The expression of N-acetylglucosaminyltransferase V (Glc-NAc-T V) is increased in many oncogenically transformed cells and in cell lines which are induced to proliferate. In order to characterize the regulation of GlcNAc-T V at the molecular level, we have examined the expression of Glc-NAc-T V mRNAs in a number of mouse tissues and in several human and rodent cell lines. The GlcNAc-T V mRNA is expressed in different amounts in the various mouse tissues, with the greatest amount observed in brain, followed by thymus, kidney, lung, intestine, heart and stomach, and no transcripts detected in liver or skeletal muscle. Measurements of GlcNAc-T V enzymatic activity, by contrast, show brain to have lower levels of activity than several of the other tissues, suggesting possible post-translational regulation. We find that there are two GlcNAc-T V transcripts in most of the RNAs analysed. The rodent cell lines all express both a 7.5 and a 9.5 kb mRNA, with the smaller transcript being more abundant. The human cells have mRNAs of 4.5 and 9.5 kb. Both mRNAs are expressed in HepG2 and MCF-7 cells, while A431 cells express only the 4.5 kb mRNA and HL60 cells express only the 9.5 kb transcript. Furthermore, only the 9.5 kb mRNA appears to be increased, along with activity, when HepG2 cells are stimulated to proliferate, suggesting that the two mRNAs may be under different regulatory controls. Also, a GlcNAc-T V-deficient, mutant lymphoma cell line, PHAR2.1, was found to express mRNAs which are larger than the normal mRNAs, possible due to an insertion or aberrant splicing, resulting in a defective mRNA.

Tissue and tumor expression of a cell surface glycoprotein complex containing an integral membrane glycoprotein activator of p185neu.

Ascites 13762 rat mammary adenocarcinoma cells contain an abundant heterodimeric cell surface glycoprotein complex. It is composed of a transmembrane subunit and a sialomucin subunit and is the product of a single gene. The transmembrane subunit has two EGF-like domains and activates the proto-oncogene receptor kinase p185neu. Southern blot comparisons of the ascites tumor and rat liver demonstrated the presence of the gene encoding the complex in normal tissues and showed an amplification of about fivefold in the ascites tumor. Polymerase chain reaction assays showed the presence of mRNA for the complex in rat brain and lung, but not in liver, pancreas, placenta, intestine, kidney, ovary and uterus. Northern blot analyses showed that the 9 kb transcript for the complex is expressed at an approximately 500-fold higher level in the ascites cells than in rat brain. Immunocytochemical studies using antiserum directed against the transmembrane subunit showed its presence in bronchial epithelium, brain ependymal and neurons of four day old animals and in the endoderm and neuronal cells of embryos. Similar immunocytochemical studies showed the presence of the transmembrane subunit in some human breast tumors. These results suggest that the gene encoding this complex is regulated in a tissue-specific manner, is overexpressed in some tumors and may play a role in tumor progression.

Modification of glycoproteins by N-acetylglucosaminyltransferase V is greatly influenced by accessibility of the enzyme to oligosaccharide acceptors.

The formation of tri- and tetraantennary complex-type N-linked oligosaccharides in animal glycoproteins is partly regulated by UDP-N-acetylglucosamine:beta-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (EC 2.4.1.155) (GlcNAc-T V), which generates 2,6-branched mannose. In Chinese hamster ovary (CHO) cells we found that 2,6-branched mannosyl structures are preferentially contained on lysosome-associated membrane proteins (LAMPs) and are generally low or absent in other cellular glycoproteins (Do, K.-Y. and Cummings, R.D. (1993) J. Biol. Chem. 268, 22028-22035). To determine the mechanism by which GlcNAc-T V appears to preferentially recognize glycoproteins, we examined the activity of purified GlcNAc-T V toward a variety of glycoprotein acceptors. Because GlcNAc-T V requires as acceptors oligosaccharides lacking outer galactosyl and sialyl residues, we utilized two classes of acceptor preparations. The first class of acceptor was enzymatically desialylated (DS) and degalactosylated (DG) preparations of bovine fetuin, human transferrin, and human fibrinogen. The second class was glycoproteins in extracts of the mutant CHO cell line, Lec8 CHO, which cannot add galactose or sialic acid to N-linked oligosaccharides. GlcNAc-T V was highly active toward DSDG-fetuin, -transferrin, and -fibrinogen (Km values ranged between 30 and 74 microM), and the catalytic efficiencies (Vmax/Km) of the enzyme toward different acceptors were comparable. In the case of fetuin, each of its three sites for attachment of N-linked oligosaccharides were shown to be utilized equally well by GlcNAc-T V. Notably, the enzyme exhibited a 2-3-fold higher rate of transfer toward DSDG-transferrin when it was further denatured by reduction and S-carboxymethylation. When extracts of Lec8 CHO cells were used as acceptors, GlcNAc-T V preferentially transferred to LAMPs, and only low level transfer was observed to other cell-derived glycoproteins, thus demonstrating specificity of GlcNAc-T V toward native glycoprotein acceptors. When the cell-derived glycoproteins were denatured by reduction and S-carboxymethylation prior to use as acceptors for Glc-NAc-T V, significant transfer occurred to other glycoproteins. These results demonstrate that the mechanism of glycoprotein-specific branching by GlcNAc-T V is determined primarily by its accessibility to available bi/triantennary oligosaccharides on glycoproteins and not by its recognition of peptide determinants or conformation-specific determinants.

Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function.

GP85 is one of the most common hemopoietic isoforms of the cell adhesion molecule, CD44. CD44(GP85) is known to contain at least one ankyrin-binding site within its 70 aa cytoplasmic domain and to bind hyaluronic acid (HA) with its extracellular domain. In this study we have mapped the ankyrin-binding domain of CD44(GP85) by deleting various portions of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that the ankyrin-binding domain resides between amino acids 305 and 355. Biochemical analyses, using competition binding assays and a synthetic peptide (NGGNGT-VEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankryin binding. Furthermore, we have constructed a fusion protein in which this 15 aa sequence of CD44(GP85) is transplanted onto another transmembrane protein which does not bind ankyrin. Our results show that this fusion protein acquires the ability to bind ankyrin confirming that the sequence (306NGGNGTVEDRKPSE320L) is a critical part of the ankryin-binding domain of CD44(GP85). In addition, we have demonstrated that deletion of this 15 aa ankyrin-binding sequence from CD44(GP85) results in a drastic reduction (> or = 90%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular matrix interactions.

Molecular cloning and sequencing of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli with sequence similarities to retroviral Gag proteins.

The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected cells exhibited fewer microfilament cables than untransfected neighboring cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a cellular protein alters the properties of the tumor cell to provide a selective advantage for tumor growth in the animal.

Molecular cloning and sequencing of the mucin subunit of a heterodimeric, bifunctional cell surface glycoprotein complex of ascites rat mammary adenocarcinoma cells.

Ascites sublines of the highly metastatic 13762 rat mammary adenocarcinoma contain abundant amounts of a heterodimeric cell surface glycoprotein complex composed of a mucin subunit ASGP-1 (ascites sialoglycoprotein-1) and a transmembrane subunit (ASGP-2). Previous studies showed that the complex is synthesized from a single polypeptide encoded by a 9 kb transcript. The sequence of the transmembrane subunit was obtained from a 5-kilobase (kb) cDNA isolated from a plasmid library (Sheng, Z., Wu, K., Carraway, K. L., and Fregien, N. (1992) J. Biol. Chem. 267, 16341-16346). Completion of the sequence of this cDNA revealed the C-terminal domain of ASGP-1, which is rich in serine and threonine but contains no typical mucin-type repeats. The remainder of the sequence of ASGP-1 and the 9-kb transcript was obtained by two 5'-RACE (rapid amplification of cDNA ends) steps and primer extension analysis. These results revealed that the 5' half of the 9-kb transcript contains a short 5'-noncoding region and encodes a signal sequence, a short nonrepeat region, and a repeat domain containing 11 repeats. Nine of these repeats are found in tandem, but the two end repeats are separated from the others by short unique sequences. The repeats vary from 117-124 amino acids and are 70-90% identical to a consensus sequence. Overall, the sequence predicts that ASGP-1 contains 2172 amino acids (M(r) 224,190), 43% of which are serine and threonine. We propose that the complex of this mucin and its transmembrane subunit, which contains growth factor-modulating activity, may play an important role in tumor progression.

Expression of the protein zero myelin gene in axon-related Schwann cells is linked to basal lamina formation.

A Schwann cell has the potential to differentiate into either a myelinating or ensheathing cell depending upon signals received from the axon that it contacts. Studies focusing on the pathway leading to myelination demonstrated that Schwann cells must form a basal lamina in order to myelinate an axon. In this report, we describe studies that indicate that initiation of basal lamina synthesis is required for Schwann cells to distinguish between myelination-inducing axons and axons that do not induce myelination, and to respond by undergoing the appropriate genetic and cellular changes. We have used high resolution in situ hybridization, immunocytochemistry and electron microscopy to examine changes in gene expression and morphology of Schwann cells differentiating into myelin-forming cells in vitro. These experiments were carried out in dorsal root ganglion neuron/Schwann cell co-cultures maintained in either serum-free, serum-only or serum-plus-ascorbate-containing medium. We have made four novel observations that contribute significantly to our understanding of how basal lamina and myelination are linked. (1) The addition of ascorbate (in the presence of serum), which promotes basal lamina production, appears to induce expression of the protein zero gene encoding the major structural protein of myelin. Moreover, expression of protein zero mRNA and protein, and its insertion into myelin membranes, occurs only in the subset of Schwann cells contacting myelination-inducing axons. Schwann cells in contact with axons that do not induce myelination, or Schwann cells that have not established a unitary relationship with an axon, do not express protein zero mRNA although they produce basal lamina components. (2) In serum-free conditions, a majority of Schwann cells express protein zero mRNA and protein, but this change in gene expression is not associated with basal lamina formation or with elongation of the Schwann cell along the axon and elaboration of myelin. (3) In the presence of serum (and the absence of ascorbate), Schwann cells again fail to form basal lamina or elongate but no longer express protein zero mRNA or protein. (4) Myelin-associated glycoprotein and galactocerebroside, two additional myelin-specific components, can be expressed by Schwann cells under any of the three culture conditions. Therefore, we have demonstrated that axonal induction of protein zero gene expression in Schwann cells is subject to regulation by both serum- and ascorbate-dependent pathways and that not all myelin-specific proteins are regulated in the same manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation, characterization, and expression of a cDNA encoding N-acetylglucosaminyltransferase V.

A cDNA clone for the complete coding sequence for alpha-1,3(6)-mannosylglycoprotein beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155) was isolated and expressed in COS-7 cells. Degenerate oligonucleotide primers for polymerase chain reaction were synthesized based on the amino acid sequence of three tryptic peptides isolated from affinity-purified GlcNAc-T V. Polymerase chain reaction amplimers were isolated from rat and mouse mRNA. A cDNA-encoding full-length enzyme was isolated from a rat 1 cell (EJ-ras-transformed) library and sequenced. Transient expression of this clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes GlcNAc-T V. Northern analysis of rat kidney mRNA revealed a single band corresponding to a length of about 7 kilobases. Sequence analysis of the cDNA clone demonstrated an open reading frame that encoded a type II membrane protein of 740 amino acids.