25 research outputs found
Mucin Structure and Function: Insights from Molecular Biology
Mucins are highly O-glycosylated glycoproteins implicated in the protection of cells from extracellular agents. Two general classes of mucins have been described: secreted and membrane. Most, but not all, mucin structures contain a central tandem repeat region which is rich in serine and threonine and is highly variable in size between different mucins; many also contain a cysteine-rich domain. Repeat sequences are often conserved within a single molecule, such as human MUC1 protein, but are poorly conserved between species, e.g. mouse and human MUC1 protein repeats. Many mucins are polymorphic due to variable numbers of repeats, and mucin transcripts are often heterogeneous. Although mucin expression is relatively tissue specific, some mucins, such as the MUC1 and MUC2 proteins, are found in multiple tissues. Moreover, a single tissue may express more than one mucin. Limited studies suggest that regulation of mucin expression is complex. Membrane mucins have been implicated in development and tumor progression, possibly by modulating cell-cell interactions. Some mucin cysteine-rich domains may also play a role in regulatingcell proliferation. Undoubtedly, future studies using recombinant DNA probes will greatly expand our understanding of these complex molecules
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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
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Molecular cloning and sequencing of the mucin subunit of a heterodimeric, bifunctional cell surface glycoprotein complex of ascites rat mammary adenocarcinoma cells
Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function.
Abstract. 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 (~NGGNGTVEDRKPSE32°L) 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 (>190%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CIM4(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular matrix interactions. T HE 85-kD mouse lymphocyte transmembrane glycoprotein, GP85 (also known as Pgp-1), is a well known T-cell differentiation antigen (66). The cDNA sequence data indicate that mouse GP85 shares 72 % aa homology with human lymphocyte homing receptor CD44 (also called GP90 Hc ~ antigen, ECMR HI, and homing cellula
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Role of Ca2+ in the regulation of hormone receptor exposure during lymphocyte activation
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Molecular cloning of the transmembrane component of the 13762 mammary adenocarcinoma sialomucin complex : a new member of the epidermal growth factor superfamily
Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.
Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA
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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
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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
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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