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C A Gabel

Publications and source records attributed to C A Gabel.

61 records · Page 4Linked to original sources
PubMed1982

Steps in the phosphorylation of the high mannose oligosaccharides of lysosomal enzymes.

The phosphomannosyl recognition marker of acid hydrolases, which mediates their translocation to lysosomes, has been shown to be synthesized in two steps. First, N-acetylglucosamine 1-phosphate is transferred to an acceptor mannose by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, resulting in a phosphate group in diester linkage between the outer N-acetylglucosamine and the inner mannose. Next, an a-N-acetylglucosaminyl phosphodiesterase removes the N-acetylglucosamine, leaving the phosphate in monoester linkage with the underlying mannose residue. This exposed phosphomannosyl residue serves as the essential component of a recognition marker which leads to binding to high-affinity receptors and subsequent translocation to lysosomes. We propose that the first enzyme in this scheme, N-acetylglucosaminylphosphotransferase, catalyses the initial, determining step by which newly synthesized acid hydrolases are distinguished from other newly synthesized glycoproteins and thus are eventually targeted to lysosomes. The absence of this enzyme activity, as in inclusion-cell (I-cell) disease and pseudo-Hurler polydystrophy, precludes the receptor-mediated targeting of newly synthesized acid hydrolases to lysosomes. As a consequence, the enzymes are secreted into the extracellular milieu.

Animals↗
PubMed1982

Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo.

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.

Acetylglucosamine↗
PubMed1979

After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos.

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.

Animals↗
PubMed1979

Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.

Animals↗