Biomedical subjects

R G Spiro

Publications and source records attributed to R G Spiro.

At least 37 records · Page 2Linked to original sources

PubMed1996

Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi.

Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.

Amino Acid Sequence↗

PubMed1996

Endoplasmic reticulum kifunensine-resistant alpha-mannosidase is enzymatically and immunologically related to the cytosolic alpha-mannosidase.

Studies were undertaken to evaluate the relationship of the recently described (S. Weng and R. G. Spiro, 1993, J. Biol. chem. 268, 25656-25663) rat liver kifunensine (KIF)-resistant mannosidase (ER mannosidase II) to the mannose-trimming enzyme of cytosol. We observed that the ER mannosidase II manifests a large number of catalytic and immunological properties similar to those of the cytosolic alpha-mannosidase, which contrast with the quite different characteristics of the KIF-sensitive enzyme (ER mannosidase I). In addition to a mutual resistance to KIF inhibition, the cytosolic enzyme and ER mannosidase II have comparable susceptibility to blocking by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, and the latter agent was found to function effectively both in vitro and in vivo. The cytosolic and ER II mannosidases were alike in specifically excising the terminal mannose of the alpha 1,6-linked chain of Man9GlcNAc to yield Man8GlcNAc isomer C; in preferentially hydrolyzing polymannose-GlcNAc1 over polymannose-GlcNAc2 substrates; and in cleaving p-nitrophenyl alpha-D-mannoside. An immunological cross-reactivity between cytosolic mannosidase (M(r) 105 kDa) and ER mannosidase II (M(r) 82 kDa), neither of which is N-glycosylated, was established, suggesting that the latter is translocated posttranslationally into the lumen of the ER compartment in which we found it to be present as a soluble protein. Since antibodies directed against a sequence near the C-terminal end of the cytosolic enzyme reacted with ER mannosidase II while those against a sequence close to the N-terminus did not, it is likely that a proteolytic cleavage of the latter segment takes place during or after translocation. The absence in ER mannosidase II of the pronounced cobalt activation of the cytosolic enzyme suggests that the portion of the polypeptide chain removed during the 105- to 82-kDa conversion includes the binding domain for this ion.

Acetylglucosamine↗

PubMed1996

Evaluation of the early processing routes of N-linked oligosaccharides of glycoproteins through the characterization of Man8GlcNAc2 isomers: evidence that endomannosidase functions in vivo in the absence of a glucosidase blockade.

Since it has become apparent that the early processing of the N-linked oligosaccharides of glycoproteins can proceed by several routes, we undertook to determine whether the isomeric nature of Man8GlcNAc2, which is the first intermediate with the potential for structural diversity, can provide information relating to the pathways utilized in various intact cultured cells as well as in the total membrane fraction derived from these cells (BW5147.3, HepG2, HL60, F-9, and MDCK). With the use of kifunensine (KIF) to block processing by Golgi mannosidase I, it could be shown that a substantial amount of Man8GlcNAc2 components in which the terminal mannose is missing in the alpha 1,3-linked and alpha 1,6-linked chain (isomers A and C, respectively) are produced, although in the absence of the inhibitor only the B-isomer, in which the mannose of the middle chain has been excised, was apparent. Our findings in vivo and in vitro suggest that the distinctive Man8GlcNAc2 product of endomannosidase (isomer A) and of ER mannosidase II (isomer C) are not evident in the absence of KIF, since they are rapidly degraded by Golgi mannosidase I, which is located in an intracellular compartment distal to the other two enzymes and itself exclusively generates the Man8GlcNAc2 isomer B. Investigations carried out in HepG2 cells indicated that glycoproteins with N-linked oligosaccharides whose processing has been blocked by KIF at the Man8GlcNac2 isomer A and C stage can nevertheless be effectively secreted. The observation that isomer A of Man8GlcNAc2 is a specific product of endomannosidase action made it possible to demonstrate the action of this enzyme in vivo without employing a glucosidase blockade and to show that a substantial amount of the deglucosylation of N-linked oligosaccharides is carried out by this enzyme.

Acetylglucosamine↗

PubMed1995

Evaluation of the cell specificity and sulfate dependence of glomerular extracellular matrix proteoglycan synthesis.

Homogeneous cultures of epithelial, endothelial, and mesangial cells from calf glomeruli were radiolabeled with [35S]sulfate in order to evaluate their capacity for the biosynthesis of the proteoglycan (PG) components present in the glomerular extracellular matrix. Although each cell type was observed to incorporate into its matrix predominantly immunologically related heparan sulfate (HS) PGs (M(r) approximately 500 kDa), endothelial and mesangial cells also deposited substantial amounts of PGs with chondroitin sulfate (CS) and dermatan sulfate (DS) chains. The limited capacity of epithelial cells to synthesize PGs other than those containing HS was also evident from the immunologically distinct components (M(r) approximately 300 kDa) shed into the medium which in contrast to those from the endothelial and mesangial cells contained no CS and only small amounts of DS glycosaminoglycans. While the matrix proteoglycan HS chains differed in length depending on cell type, they were similar in containing the six mono- and disulfated disaccharide species previously found in bovine glomerular basement membrane, including the distinctive iduronic-GlcNSO3 (3-SO4) sequences. While the addition of sulfate to medium free of this ion brought about no change in HS PG production by any of the three cell types and the formation of CS and DS chains by epithelial and mesangial cells was unaffected, the formation of CS/DS PGs by endothelial cells was altered to a pronounced extent through the conversion of an undersulfated PG to a more polyanionic molecule. Our findings are consistent with the concept that the glomerular extracellular matrix is made up of two biosynthetically distinct regions (mesangium and basement membrane) and are relevant to an understanding of various diseases affecting the renal filter.

Animals↗

PubMed1995

Glucose entry into rat mesangial cells is mediated by both Na(+)-coupled and facilitative transporters.

Since previous studies from our laboratory have demonstrated that increased glucose consumption by cultured rat mesangial cells is accompanied by an accelerated production of type IV and type VI collagen, we have now examined the manner by which glucose is transported into these cells. A progressive stimulation of glucose uptake by the mesangial cells was observed with increasing concentrations of NaCl so that at 145 mmol/l about twice as much glucose entered the cells as in its absence (substituted by choline chloride). Moreover, since phlorizin inhibited the NaCl-promoted uptake of glucose and this salt was found to increase the accumulation of alpha-methylglucoside in a manner which could not be duplicated by KCl or mannitol, both Na(+)-coupled and facilitative glucose transporters appeared to be present in the cells. Km values of 1.93 mmol/l and 1.36 mmol/l were determined for the co-transport and facilitated transport pathways, respectively, with their Vmax being 29.5 and 18.0 nmol.mg protein-1.h-1. Both uptake activities were found to be down-regulated by exposure of the cells to high glucose and furthermore the Na(+)-dependent transport could no longer be detected after about 12 passages of the cells. Hybridization of mesangial cell mRNA with cDNA probes revealed transcripts for the Na+/glucose co-transporter as well as GLUT1 and to a lesser extent GLUT4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis.

The intracellular site for the degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis has been studied by permeabilizing the plasma membrane of metabolically radiolabeled HepG2 cells with streptolysin O. This pore-forming agent permitted us to examine the breakdown in both the cytosolic and vesicular compartments of the previously recognized (Anumula, K. R., and Spiro, R. G. (1983) J. Biol. Chem. 258, 15274-15282) polymannose components terminating in a di-N-acetylchitobiose sequence (OS-Glc-NAc2) or a single N-acetylglucosamine residue (OS-Glc-NAc1) residue. Pulse-chase studies indicated that although the OS-GlcNAc2 saccharides were about equally distributed between vesicles and cytosol and rapidly disappeared after reaching the Man8 stage, the OS-GlcNAc1 species were found predominantly in the extravesicular compartment and there underwent a distinctive demannosylation sequence resulting in the formation of a Man5GlcNAc isomer (Man alpha 1-->2Man alpha 1-->2Man alpha 1-->3(Man alpha 1-->6)Man beta 1-->4GlcNAc) which was different from the product of Golgi processing enzymes. Further trimming of this cytosolic limit product required its translocation into a vesicular compartment, believed to be lysosomes, in which Man2-4GlcNAc components appeared as the metabolic chase progressed. The accumulation of Glc1Man5GlcNAc in the cytosol during the chase suggested that glucose interferes with the cytosolic-vesicular transfer and this became even more evident by the pronounced pile-up of extravesicular Glc3Man5GlcNAc when the cells were incubated in the presence of castanospermine. Although the biological significance and mechanism of free polymannose oligosaccharide entry into the cytosol is not yet known, the possibility that it may reflect an endoplasmic reticulum-situated degradative process of glycoproteins merits consideration.

Bacterial Proteins↗

PubMed1994

Ligand affinity chromatographic purification of rat liver Golgi endomannosidase.

In order to achieve isolation of endo-alpha-D-mannosidase, a Golgi-located processing enzyme that accomplishes deglucosylation of glycoproteins with N-linked carbohydrate units by cleaving the linkage between the glucose-substituted mannose residue and the remainder of the oligosaccharide, we have prepared an affinity matrix (Glc alpha 1-->3Man-O-(CH2)8CONH-Affi-Gel 102) containing the derivative of the characteristic disaccharide product of this enzyme. Chromatography of a Triton extract of rat liver Golgi membranes on a column of this gel in the presence of castanospermine to prevent binding of alpha-glucosidases permitted a rapid purification of the endomannosidase (70,000-fold over the homogenate) with a 12% yield. This purified enzyme was free of other processing glycosidases and was completely inhibited by Glc alpha 1-->3(1-deoxy)mannojirimycin. Examination of the endomannosidase by SDS-polyacrylamide gel electrophoresis revealed a doublet (M(r) 60,000 and 56,000) with the bands being of approximately equal density. Gel permeation high performance liquid chromatography indicated that in its native form the enzyme has an oligomeric structure (M(r) approximately 560,000) consisting of eight to ten subunits.

Animals↗

PubMed1994

Synthesis of type VI collagen by cultured glomerular cells and comparison of its regulation by glucose and other factors with that of type IV collagen.

Homogeneous cultures of calf glomerular mesangial and endothelial cells were found to be active in the synthesis of type VI as well as type IV collagen in contrast to the epithelial cells that were devoted primarily to the production of the latter collagen. Studies with rat mesangial cells indicated that they responded to high glucose (20 mM) in the medium by a significant (P < 0.001) increase in type VI collagen synthesis as measured by the production of the protein and its mRNA level, both of which were closely correlated to each other and to glucose consumption. Similar observations were made with type IV collagen, but the enhanced formation of this protein was not as rapidly apparent as that of type VI and, moreover, could not be as readily reversed on restoration of the glucose to a physiological level (5 mM). Evaluation of a number of other agents indicated that although mannitol had no effect, L-glucose and NaCl significantly stimulated synthesis of both type VI and IV collagens and glucose consumption. Insulin-like growth factor I and aldosterone, on the other hand, also increased glucose consumption but brought about an enhancement of only type IV collagen production, suggesting that the two collagens are independently regulated. This possibility was supported by our observation that pyruvate, which was actively taken up by the cells, selectively stimulated type IV collagen production.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗

PubMed1993

Demonstration that a kifunensine-resistant alpha-mannosidase with a unique processing action on N-linked oligosaccharides occurs in rat liver endoplasmic reticulum and various cultured cells.

A novel alpha-mannosidase has been identified in rat liver endoplasmic reticulum (ER) which at neutral pH processes the Man9GlcNAc oligosaccharide of glycoproteins by specifically cleaving the terminal mannose residue of the alpha 1,6-linked chain to yield Man8GlcNAc, isomer C. This enzyme accounted for about half of the total ER alpha-mannosidase activity and was fully active at the concentration (0.25 microM) of kifunensine (KIF) completely inhibitory to the action of the ER enzyme which by removing the terminal sugar of the middle chain converts Man9GlcNAc to Man8GlcNAc isomer B; both ER enzymes, however, were inhibited in a similar manner by 1-deoxymannojirimycin (IC50 = 0.2 mM) and their action could not be distinguished with this agent. The KIF-resistant mannosidase which functioned optimally in the presence of 0.1-0.5% Triton X-100 did not show the high susceptibility to EDTA demonstrated by the KIF-sensitive enzyme and unlike the latter had the capacity to hydrolyze p-nitrophenyl-alpha-D-mannoside (Km = 0.45 mM); it had no specific cation requirements, but its activity was greatly reduced in the presence of Zn2+. In isolated ER membranes as well as in intact carbonyl cyanide m-chlorophenylhydrazone-treated cells, the processing pattern was substantially different in the presence of KIF than in its absence; while in the latter instance Man9GlcNAc was readily converted to Man6GlcNAc, the KIF-resistant enzyme was limited in its capacity to go beyond Man8GlcNAc. The KIF-resistant alpha-mannosidase was found in substantial amounts in all cell lines examined (HL-60, BW5147.3, MOLT-4, K-562, HepG2, Chinese hamster ovary, F-9, Madin-Darby canine kidney, FRTL-5). The finding that mannose removal from N-linked oligosaccharides can be initiated in two distinctive manners substantially broadens our concept of the processing events which can occur before a glycoprotein reaches the Golgi complex or to which ER resident molecules can be exposed.

1-Deoxynojirimycin↗

PubMed1993

Characterization of endomannosidase inhibitors and evaluation of their effect on N-linked oligosaccharide processing during glycoprotein biosynthesis.

Endo-alpha-D-mannosidase is a Golgi-located processing enzyme that achieves deglucosylation of N-linked carbohydrate units through its unique property of cleaving the oligosaccharide chain internally with the release of glucose-substituted mannose (Glc1-3Man). By chemically modifying the characteristic disaccharide product, Glc alpha 1-->3Man, a number of potent inhibitors of the endomannosidase were obtained, foremost among which were Glc alpha 1-->3(1-deoxy)mannojirimycin (Glc alpha 1-->3DMJ) and Glc alpha 1-->3(1,2-dideoxy)mannose (IC50 = 1.7 and 3.8 microM, respectively), which, while blocking the in vitro action of the enzyme, had negligible effect on other endoplasmic reticulum- and Golgi-processing glycosidases. Although preparation of a large number of Glc alpha 1-->3DMJ derivatives did not yield a more effective endomannosidase inhibitor it provided valuable information relating to the structural requirements for the enzyme-substrate interaction. Glc alpha 1-->3DMJ was found to be active not only on rat liver endomannosidase but also on the enzyme from a number of other sources including mouse lymphoma (BW5147.3), HepG2, baby hamster kidney, and Madin-Darby canine kidney cell lines. When tested in vivo in lymphoma and Madin-Darby canine kidney cells during a castanospermine-imposed glucosidase blockade, Glc alpha 1-->3DMJ interrupted the endomannosidase processing pathway as evident from a concomitant inhibition of complex oligosaccharide formation and Glc3Man release; similarly the capacity of the glucosidase II-deficient mouse lymphoma cell line (PHAR2.7) to synthesize complex oligosaccharides was blocked by Glc alpha 1-->3DMJ. Endomannosidase could not be detected in Chinese hamster ovary cells by in vitro assay and consistent with this these cells produced only glucosylated polymannose N-linked oligosaccharides during glucosidase blockade. It would appear that by acting in conjunction with a glucosidase inhibitor, Glc alpha 1-->3DMJ and related endomannosidase-blocking agents could have the potential of influencing the exit of glycoproteins from the endoplasmic reticulum and interfering with viral replication.

1-Deoxynojirimycin↗

PubMed1993

Inhibition of glucose trimming by castanospermine results in rapid degradation of unassembled major histocompatibility complex class I molecules.

The CMT-cKd1 cell line provides a system for studying the initial processing steps of N-linked oligosaccharides as these cells have been shown to produce major histocompatibility complex (MHC) class I molecules which, due to a defect in assembly, recycle between the endoplasmic reticulum and a pre-Golgi compartment, failing to reach the cell surface (Hsu, V.W., Yuan, L. C., Nuchtern, J. G., Lippincott-Schwartz, J., Hämmerling, G. J., and Klausner, R. D. (1991) Nature 352, 441-444). In the present study we observed that when the MHC class I heavy chain of these CMT cells was pulse-radiolabeled with [35S]methionine in the presence of the glucosidase inhibitor, castanospermine (CST), it underwent a rapid degradation during a 60-min chase, in contrast to control cells in which it remained stable during that period. The CST-promoted instability of the MHC molecule appeared to be specific, as it did not occur when 1-deoxymannojirimycin, an inhibitor of mannosidase, was added to the cells. Although endomannosidase was found to be present in the CMT cells, the electrophoretic mobility of the MHC heavy chain produced in the presence of CST indicated that deglucosylation through the alternate route provided by this enzyme did not occur. Furthermore, gamma-interferon did not prevent the rapid disappearance of the MHC molecule, although it brought about entry of this glycoprotein into the secretory pathway in cells incubated without CST. The results of our studies suggest that retention of glucose on N-linked oligosaccharides may under certain circumstances provide a signal for pre-Golgi protein degradation.

1-Deoxynojirimycin↗

PubMed1993

Effect of high glucose on type IV collagen production by cultured glomerular epithelial, endothelial, and mesangial cells.

Immunochemical and metabolic radiolabeling procedures revealed that homogeneous cultures of calf glomerular epithelial, endothelial, and mesangial cells actively synthesize type IV collagen (primarily as alpha 1 (IV)3) which is secreted into the medium and incorporated into the extracellular matrix. Exposure of confluent cultures of the three cell types to a high glucose concentration (30 mM) for 60 h resulted in a pronounced increase (two- to threefold) in type IV collagen production over that observed at a physiological level (5 mM) of this sugar, as determined by either immunoblotting or fluorography of electrophoretically separated media or cell-matrix components. The elevated glucose did not bring about a change in the rate of cell proliferation or fibronectin production. Moreover, studies with mannitol indicated that the stimulation of type IV collagen synthesis was not a function of hyperosmolarity. In contrast to the glomerular cells, glucose-induced enhancement of formation of this collagen was not observed in 3T3 cells despite a substantial acceleration in the consumption of this sugar. Time studies indicated that the response of the glomerular cells to high glucose occurs over an extended period (maximal at approximately 78 h) and, furthermore, that the stimulatory effect on type IV collagen production is only slowly reversed after restoration of the glucose to a normal level. We believe that these findings are relevant to an understanding of the sequence of events that lead to the development of diabetic glomerular lesions.

Animals↗

PubMed1992

Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells.

Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.

1-Deoxynojirimycin↗

PubMed1992

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.

1-Deoxynojirimycin↗

PubMed1992

Diabetic glomerulosclerosis--immunogold ultrastructural studies on the glomerular distribution of type IV collagen and heparan sulphate proteoglycan.

We have undertaken an ultrastructural immunogold investigation of the distribution of type IV collagen and heparan sulphate proteoglycan (HSPG) in glomeruli from the kidneys of one normal control and three patients with diabetes mellitus and proteinuria. The sample included both diffuse and nodular diabetic glomerulosclerosis. In the control and diabetic kidneys, the type IV collagen was present predominantly on the endothelial aspect of the glomerular basement membrane (GBM), and by contrast the HSPG was found mainly on the epithelial side. In the mesangium in both control and diabetic glomeruli, type IV collagen was found predominantly in the central regions, while HSPG was mostly restricted to the region beneath the epithelial cells. Consequently, where there is a marked increase in mesangial matrix with nodule formation in diabetics there is a corresponding increase in the amount of type IV collagen but not of HSPG. Although the three diabetic patients were proteinuric, the HSPG was not decreased in the thickened GBMs.

Adult↗

PubMed1992

Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives.

A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.

Chromatography, High Pressure Liquid↗

PubMed1991

Localization and structure of the asparagine-linked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule.

Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.

Animals↗

PubMed1991

Characterization of heparan sulfate proteoglycan from calf lens capsule and proteoglycans synthesized by cultured lens epithelial cells. Comparison with other basement membrane proteoglycans.

After extraction with 4 M guanidinium chloride and purification by DEAE-cellulose chromatography, the heparan sulfate proteoglycan (HSPG) of calf anterior lens capsule was found to consist of two immunologically related components (Mr = 340,000 and 250,000) which upon deglycosylation with trifluoromethanesulfonic acid yielded core proteins with Mr values of 170,000 and 145,000. The heparan sulfate chains were uniform in size (Mr = 14,000) and manifested a clustering of sulfate groups in a peripheral domain. From the decrease in Mr observed after heparitinase digestion, it could be estimated that 6 and 11 glycosaminoglycan chains were present in the Mr = 250,000 and 340,000 components respectively. The occurrence of N-linked oligosaccharides was evident from the size difference of the heparitinase- and trifluoromethane-sulfonic acid-treated proteoglycans (approximately 20 kDa), as well as from the presence of a substantial number of mannose residues; furthermore, interaction of the capsule proteoglycan with Bandeiraea simplicifolia I suggested that these carbohydrate units contains terminal alpha-D-Gal groups. Cultured lens epithelial cells deposited a single [35S]sulfate-labeled proteoglycan into their matrix (Mr = 400,000) which was immunologically related to the lens capsule proteoglycan and contained only heparan sulfate chains. In addition to this component, the medium from these cells contained an immunologically unrelated HSPG (Mr = 150,000) as well as a chondroitin sulfate proteoglycan (Mr = 240,000). Examination of bovine glomeruli indicated that, in addition to the previously described 200-kDa HSPG, an immunologically related 350-kDa component was also present. This size heterogeneity, which is comparable to that seen in the lens capsule, is most readily attributable to proteolytic processing of a precursor molecule. Studies with polyclonal antibodies demonstrated only limited cross-reactivities between the Engelbreth-Holms-Swarm proteoglycan and the components from lens capsule and glomerular basement membrane; since even the latter two differed somewhat in their antigenic sites, it would appear that cell- and species-dictated genetic differences as well as post-translational events contribute to the diversity observed in basement membrane HSPGs.

Amino Acids↗