Browsing by Author "Gorbea, Carlos M."

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    Expression and glycosylation of meprin isoforms
    Gorbea, Carlos M. (Virginia Tech, 1992-09-15)
    Meprin A and meprin B are disulfide-linked, oligomeric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (α subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (β subunits) in situ, and the enzyme from C3H/He mice, a strain that does not express α subunits, has been characterized. Evidence from this and previous studies indicate that β subunits are expressed in all mouse strains. Meprins were characterized with regard to their glycosylation by lectin blotting. Both meprin A and meprin B bound the lectins concanavalin A and the erythroagglutinin from Phaseolus vulgaris indicating that both enzymes contain high mannose and bisected biantennary complex type oligosaccharides. However, meprin A, but not meprin B, bound the agglutinins from Ricinus communis, Datura stramonium, and the leukoagglutinin from Phaseolus vulgaris, indicating that complex-type N-glycosylation differs in these proteinases. Lectin blots of membrane proteins from C57BL/6 mice indicated that there were differences between adult male and female mice in the glycosylation (specifically in the complex type oligosaccharides) of the α subunit of meprin A. A marked degree of carbohydrate heterogeneity was observed in meprin A from males as compared to the enzyme isolated from female mice. Additionally, the data indicated that at least three of the ten potential glycosylation sites in the meprin α subunit are glycosylated. Overall, these studies expand our understanding of how estrogens affect glycosylation of meprin A. The oligomeric organization of the meprins was examined in brush border membrane fractions from a random-bred strain (lCR) and two inbred strains of mice (C57BL/6 and C3H/He). The random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-P AGE) in the absence of reducing agents. The subuβnits in all three oligomers were linked by disulfide bridges. Western blotting using anti-α monoclonal antibodies revealed that α subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with polyclonal antibodies specific for the β subunit (110 kDa) revealed the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of α and β subunits, respectively, and that the 440-kDa complex is a heterotetramer composed of disulfide-linked α and subunits. C57BL/6 mice expressed both α and β subunits and contained tetramers composed of α₄ and α₂β₂. C3H1He mice expressed only the 110-kDa β subunits and the β₄ oligomer. This type of multimeric organization of covalently-linked subunits is unique for the known endopeptidases. Initial cloning of the mouse meprin β subunit revealed that the enzyme belongs to the recently described astacin family of metalloendopeptidases. The β subunit polypeptide had a molecular mass of 88 kDa as determined after SDSPAGE of brush border membrane proteins treated with glycosidases. Nucleotide sequencing, internal peptide sequences from the β subunit, and NH₂-terminal sequence analyses (39 residues) indicated that at the amino acid sequence level, mouse β is approximately 55 % identical to mouse , and 85 % identical to the rat β subunit. These and other studies indicate that α and β are closely related products of divergent evolution. Northern blot analyses of different tissues from C57BL/6 and C3H/He mice indicate that β subunit mRNA can be detected in kidney and intestine, in contrast to the α subunit which is only present in kidney tissue. Initial studies in mouse intestinal brush border membranes indicated that the characteristic latency of kidney β subunits may be absent in the intestinal enzyme. This observation may reflect activation of the mouse β subunit by trypsin in the intestinallumen. The activation of β in the kidney by trypsin-like proteinases is reminiscent of the activation of protein zymogens and may serve as a means of regulation of the proteolytic activity of the proteinase at the cell surface.
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    Glycolipids in mouse F9 teratocarcinoma cells: some changes associated with retinoic acid-induced differentiation
    Gorbea, Carlos M. (Virginia Tech, 1991-01-15)
    To investigate the changes in glycolipid biosynthesis during early embryogenesis mouse F9 teratocarcinoma cells were induced to differentiate in vitro in the presence of retinoic acid. Control embryonal carcinoma cells and their differentiated derivatives, RNF9 cells, were metabolically-radiolabeled with [6-3H]galactose or [6-3H]glucosamine, and their glycolipids were compared. The neutral and acidic glycolipid fractions from both cell lines were subjected to ozonolysis and alkali fragmentation or endoglycoceramidase digestion to release the glycolipid-derived oligosaccharides. The neutral oligosaccharides were separated according to size by gel filtration and high performance liquid chromatography. These analyses indicated that differentiated F9 cells synthesized less high molecular weight oligosaccharides (containing more than 5 sugar residues) relative to controls. Serial lectin affinity chromatography on columns of immobilized Helix pomatia. Wisteria f1oribunda. Griffonia simplicifolia-I and Ricinus communis-' agglutinins followed by reduction and permethylation revealed that globoside (GaINAcβ1, 3Galα1, 4Galβ1, 4Glc) and lactose (Galβ1,4Glc) are the principal glycolipid-derived oligosaccharides synthesized by F9 and RNF9 cells. An increased biosynthesis of these components was observed in RNF9 cells relative to controls. These changes paralleled the reduced biosynthesis of Forssman pentasaccharide (GaiNAcα 1 ,3GaINAcβ1 ,3Galα1 ,4Galβ1 ,4Glc) reported previously. Normalization of the incorporation of 3H-monosaccharides in glycolipid-derived oJigosaccharides to the number of cells indicated a 2-6 fold increase in the incorporation of radioactive precursors in RAlF9 cells relative to F9 controls, suggesting that an enhancement in glycosphingolipid biosynthesis accompanies the differentiation of F9 cells. The monosialylganglioside-derived oligosaccharides obtained from F9 and RAlF9 cells were separated by anion exchange chromatography. A reduced biosynthesis of high molecular weight components was observed in RAlF9 cells when compared with undifferentiated F9. Lectin affinity chromatography on immobilized Maackia amurensis agglutinin followed by reduction and permethylation indicated a dramatic increase in the synthesis of GM1 (Galβ1,3Ga1NAcβ1,4[NeuAcα2,3] Galβ1,4Glc) and GM3 (NeuAcα2,3Galβ1,4Glc) in RAlF9 cells relative to controls. These changes were accompanied by a decrease in the synthesis of sialyltetrasaccharide a (NeuAcα2,3Galβ1 ,3GlcNAcβ1 ,3Galβ1,4Glc) and sialylparagloboside (NeuAcα2,3Galβ1 ,4GlcNAβ1 ,3Galβ1 ,4Glc) in the differentiated cells. These observations are in agreement with previous reports in leukemic and human embryonal carcinoma cell lines and may be related to the growth arrest and antigenic changes associated with F9 differentiation. In the work reported herein, serial lectin affinity chromatography in concert with permethylation analysis prove to be powerful methods for the isolation and characterization of glycolipid-derived oligosaccharides. The application of these methods has allowed the unequivocal identification of main glycosphingolipid components as well as of some representing less than 1 % of the total glycolipids synthesized by two cell lines. This information should provide the basis for further studies involving glycosyltransferas.