Release of polymannose oligosaccharides from vesicular stomatitis virus G protein during endoplasmic reticulum-associated degradation.

To further explore the localization of the N-deglycosylation involved in the endoplasmic reticulum (ER)-associated quality control system we studied HepG2 cells infected with vesicular stomatitis virus (VSV) and its ts045 mutant, as in this system oligosaccharide release can be attributed solely to the VSV glycoprotein (G protein). We utilized the restricted intracellular migration of the mutant protein as well as dithiothreitol (DTT), low temperature, and a castanospermine (CST)-imposed glucosidase blockade to determine in which intracellular compartment deglycosylation takes place. Degradation of the VSV ts045 G protein was considerably greater at the nonpermissive than at the permissive temperature; this was reflected by a substantial increase in polymannose oligosaccharide release. Under both conditions these oligosaccharides were predominantly in the characteristic cytosolic form, which terminates in a single N-acetylglucosamine (OS-GlcNAc(1)); this was also the case in the presence of DTT, which retains the G protein completely in the ER. However when cells infected with the VSV mutant were examined at 15 degrees C or exposed to CST, both of which represent conditions that impair ER-to-cytosol transport, the released oligosaccharides were almost exclusively (> 95%) in the vesicular OS-GlcNAc(2) form; glucosidase blockade had a similar effect on the wild-type virus. Addition of puromycin to glucosidase-inhibited cells resulted in a pronounced reduction (> 90%) in oligosaccharide release, which reflected a comparable impairment in glycoprotein biosynthesis and indicated that the OS-GlcNAc(2) components originated from protein degradation rather than hydrolysis of oligosaccharide lipids. Our findings are consistent with N-deglycosylation of the VSV G protein in the ER and the subsequent transport of the released oligosaccharides to the cytosol where OS-GlcNAc(2) to OS-GlcNAc(1) conversion by an endo-beta-N-acetylglucosaminidase takes place. Studies with the ts045 G protein at the nonpermissive temperature permitted us to determine that it can be processed by Golgi endomannosidase although remaining endo H sensitive, supporting the concept that it recycles between the ER and cis-Golgi compartments.

Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15 degrees C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus.

Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases.

The occurrence of sulfate substituents on several positions of glycoprotein N-linked oligosaccharides prompted us to determine the subcellular localization and temporal relationships of the addition of these anionic groups employing as a model system the hemagglutinin (HA) produced by influenza virus-infected Madin-Darby canine kidney (MDCK) cells. It became apparent from a study of the HA glycoprotein in subcellular fractions resolved by Nycodenz gradient centrifugation following pulse-chase radiolabeling that sulfation of the complex N-linked oligosaccharides occurs only after they have been processed to an endo-beta-N-acetylglucosaminidase-resistant state and have reached the medial/trans Golgi and the trans Golgi network (TGN), with the former carrying out most of the sulfation activity. Hydrazine/nitrous acid/NaBH(4) treatment of the HA from the subcellular fractions indicated that C-3 of the galactose as well as C-6 of the N-acetylglucosamine residues of the N-acetyllactosamine chains became sulfated in these post ER fractions, as did the C-6 of the outer N-acetylglucosamine of the di-N-acetylchitobiose core. Consistent with the specificities of the stepwise assembly of the N-acetyllactosamine branches, we observed that the 3'-phosphoadenosine 5'-phosphosulfate (PAPS):GlcNAc-6-O-sulfotransferase migrated in the gradient to a medial/trans Golgi position while in contrast the PAPS:Gal-3-O-sulfotransferase was found in both Golgi and TGN locations. In accordance with the concept that beta-galactosylation must precede the sulfation catalyzed by the latter enzyme, we observed the presence of UDP-Gal:GlcNAc galactosyltransferase in both these sites in the MDCK cells. The presence of the Gal-3-O-sulfotransferase in the TGN is particularly important in the influenza virus-infected cells, as it makes possible the addition of terminal anionic groups after removal of the sialic acid residues by the viral neuraminidase.

Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors.

Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc(1-3)Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of mono-glucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-beta-N- acetylglucosaminidase H can distinguish between those terminating in a single N-acetylglucosamine or in a di-N-acetylchitobiose sequence.

Immunohistochemical evaluation of endomannosidase distribution in rat tissues: evidence for cell type-specific expression.

Asparagine-linked oligosaccharides of glycoproteins are subject to a series of trimming reactions by glucosidases and mannosidases in the endoplasmic reticulum which result in the removal of all three glucose residues and several of the nine mannose residues. At present, endomannosidase represents the only processing enzyme which cleaves internally and provides an alternate deglucosylation pathway. However, in contrast to the endoplasmic reticulum residential proteins glucosidase I and II, endomannosidase is primarily situated in the Golgi apparatus of rat liver hepatocytes and hepatocyte cell lines. We have performed a confocal immunohistochemical study to investigate endomannosidase in various rat tissues and used a monoclonal antibody against Golgi mannosidase II as a marker for the Golgi apparatus. Although immunofluorescence for both endomannosidase and Golgi mannosidase II was detectable in the epithelia of many tissues, renal proximal tubular cells, cortex and medulla of adrenal gland, gastric mucosa, and Leydig cells of testis were unreactive for endomannosidase. Furthermore, the endothelia in all studied tissues were unreactive for endomannosidase but positive for Golgi mannosidase II. It is concluded that by immunohistochemistry endomannosidase exhibits a cell type-specific expression in rat tissues.

Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum.

Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact sites between the two organelles. Furthermore, the presence of this glycoprotein in at least two major mitochondrial multienzyme complexes would be consistent with a role in mitochondrial translocations.

Molecular cloning and expression of rat liver endo-alpha-mannosidase, an N-linked oligosaccharide processing enzyme.

A clone containing the open reading frame of endo-alpha-D-mannosidase, an enzyme involved in early N-linked oligosaccharide processing, has been isolated from a rat liver lambdagt11 cDNA library. This was accomplished by a strategy that involved purification of the endomannosidase from rat liver Golgi by ligand affinity chromatography (Hiraizumi, S., Spohr, U., and Spiro, R. G. (1994) J. Biol. Chem. 269, 4697-4700) and preparative electrophoresis, followed by sequence determinations of tryptic peptides. Using degenerate primers based on these sequences, the polymerase chain reaction with rat liver cDNA as a template yielded a 470-base pair product suitable for library screening as well as Northern blot hybridization. EcoRI digestion of the purified lambda DNA released a 5.4-kilobase fragment that was amplified in Bluescript II SK(-) vector. Sequence analysis indicated that the deduced open reading frame of the endomannosidase extended from nucleotides 89 to 1441, encoding a protein of 451 amino acids and corresponding to a molecular mass of 52 kDa. Data base searches revealed no homology with any other known protein. When a vector coding for this protein fused to an NH2-terminal peptide containing a polyhistidine region was introduced into Escherichia coli, high levels of the enzyme were expressed upon induction with isopropyl-beta-D-thiogalactoside. Purification of the endomannosidase to electrophoretic homogeneity from E. coli lysates was accomplished by Ni2+-chelate and Glcalpha1-->3Man-O-(CH2)8CONH-Affi-Gel ligand chromatographies. Polyclonal antibodies raised against this protein reacted with Golgi endomannosidase. By both immunoblotting and silver staining, the purified E. coli-expressed enzyme was approximately 8 kDa smaller than anticipated from the open reading frame; timed induction studies indicated that this was due to scission of the enzyme's COOH-terminal end by host cell proteases. All rat tissues examined demonstrated mRNA levels (4.9-kilobase message) for the endomannosidase that correlated well with their enzyme activity.

Suspension culture of differentiated rat heart myocytes on non-adhesive surfaces.

Cardiac myocytes isolated from adult rat ventricles have been maintained in a stable, differentiated state for prolonged periods by the use of suspension culture on hydrophobic tissue culture inserts or agarose-coated plates. The success of this procedure depends on the use of low-serum media to prevent myocyte-myocyte interaction and proliferation of any residual endothelial cells. Myocytes cultured in this manner retain many of their structural characteristics, suggesting that maintenance of their elongated irregular shape is not dependent on interaction with extracellular matrix. They also exclude trypan blue, can be vitally stained by the uptake and reduction of the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], synthesize myosin and when returned to adhesive surfaces are capable of attachment and attendant dedifferentiation. Stability of the myocytes in suspension permits their use in co-culture experiments; specifically, myocytes separated from endothelial cells by the hydrophobic membrane of the tissue culture insert stimulated proliferation of the latter cells, suggesting this to be a useful system for studying myocyte-endothelial cell interaction.

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.

Isolation of rat heart endothelial cells and pericytes: evaluation of their role in the formation of extracellular matrix components.

In order to facilitate investigation of the cells responsible for overproduction of type VI collagen in the extracellular matrix surrounding the capillaries of diabetic rat myocardium, procedures have been developed for the isolation from this tissue of endothelial cells as well as a cell type identified as pericytes. This was accomplished by enzymatic and mechanical disruption of ventricles from young rats (125 g) followed by removal of myocytes through their nonadherence to tissue culture surfaces. Endothelial cells were separated by fluorescence-activated cell sorting after staining with rhodamine-labeled acetylated low density lipoprotein and were identified by their monolayer growth pattern, reaction with anti-von Willebrand factor and the ability to form capillary-like tubes induced by low serum concentration. Pericytes were purified by selective scraping for removal of other cell types and were identified by their irregular shape, overlapping growth pattern at confluence, reaction with anti-smooth muscle actin and content of GLUT4 glucose transporter. Fibroblasts, visualized after staining with rhodamine-labeled alpha 2-macroglobulin, were only rarely detected. Analysis of collagen by immunoblotting indicated formation by both cell types of alpha 1(IV) collagen as well as the three subunits of type VI (alpha 3 at 205 kDa and alpha 1 plus alpha 2 at 150 kDa). Both endothelial cells and pericytes demonstrated transcripts for types VI, IV and I collagen, as well as fibronectin, but while the level of the mRNA for type IV collagen was higher in pericytes than in endothelial cells, the reverse was true for collagens VI and I and fibronectin. These observations suggest that both endothelial cells and pericytes contribute to formation of the myocardial capillary matrix, but that changes involving only type VI collagen, such as occur in diabetic cardiomyopathy, may reflect a response primarily of endothelial cells.

Effect of high glucose on formation of extracellular matrix components by cultured rat heart endothelial cells.

In an attempt to define the basis for the microvascular changes observed in diabetic myocardium, a study was undertaken on the effect of elevated glucose on the synthesis by rat heart endothelial cells of the extracellular matrix components, types VI, IV and I collagen, as well as fibronectin. Confluent cultures of these cells, isolated by fluorescence-activated cell sorting after treatment with rhodamine-labelled acetylated low density lipoprotein, showed a three to fivefold enhancement in the synthesis of type VI collagen after exposure for 48 h to high glucose (20 to 30 mmol/l), as determined by immunoblot analysis. Increased production of type IV collagen and fibronectin was also observed, but the change was smaller and no effect on type I collagen was found. Measurement of mRNA levels by hybridization with cDNA probes indicated that 48-h exposure to high glucose significantly increased the level of transcripts for type VI and IV collagens but not for type I collagen. While glucose consumption by endothelial cells in high glucose doubled in the initial 24-h period, utilization returned to normal by 48 h, concomitant with a reduction in GLUT1 transcript levels, suggesting that signals for stimulation of collagen synthesis must be active during the initial period of exposure to elevated glucose levels.

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)

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)

Increased rat myocardial type VI collagen in diabetes mellitus and hypertension.

Diabetic cardiomyopathy, a condition characterized by the accumulation of carbohydrate-containing material surrounding the myocardial small blood vessels, has been studied in alloxan-diabetic normotensive and hypertensive rats. Immunochemical techniques were used to monitor several extracellular matrix constituents present in extracts of cardiac tissue, namely types I, IV and VI collagen, laminin and fibronectin, as well as myosin. These studies have indicated that after induction of diabetes, type VI collagen but none of the other matrix components studied, was significantly increased (from 2.29 +/- 0.04 mg/g in normal to 2.85 +/- 0.18 mg/g in diabetic ventricles, p < 0.01). Hypertension, whether induced by the clipping of one renal artery or genetically determined (spontaneously hypertensive rats), resulted in a similar elevation in type VI collagen (2.71 +/- 0.12 mg/g, p < 0.005 compared to normal rats). In the presence of diabetes plus hypertension the effect was not additive, the type VI collagen level being 2.93 +/- 0.15 (p < 0.001 compared to normal rats). Basement membrane collagen (type IV) in the myocardium appeared to be unaffected by diabetes or hypertension and the myosin contents of the hearts of the four experimental groups were similar. Quantitative determinations indicate that compared to type IV collagen, laminin or fibronectin, type VI collagen represents the major periodic acid-Schiff-reactive extracellular constituent of the rat ventricle. Its preferential increase in the heart in diabetes may provide insight into the molecular mechanisms of the diabetic microvascular disease.

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.

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.

Myocardial glycoproteins in diabetes: type VI collagen is a major PAS-reactive extracellular matrix protein.

An investigation of myocardial glycoproteins was undertaken to elucidate the molecules responsible for the periodic acid-Schiff (PAS) reactivity of the increased extracellular matrix of diabetic cardiomyopathy. Perfusion with radiolabeled mannose indicated an enhanced formation of matrix components in the diabetic compared to the normal rat heart. Electrophoretic separation of radiolabeled extracts demonstrated the presence of glycoproteins with Mr values of 205, 142 and 90 kDa which could be separated by Bio-Gel A-5 m filtration. Fractionation of non-perfused hearts resulted in the isolation of only the 205 and 142 kDa components, which were shown by amino acid analyses and collagenase digestion to belong to the collagen family of proteins and by immunoblotting to represent type VI collagen. The carbohydrate content of these rat myocardial type VI collagen subunits, determined from monosaccharide analyses, was 11 and 12%, respectively, and N-glycanase digestion of the 142 kDa chain resulted in a decrease in size of approximately 14 kDa, indicating the presence of asparagine-linked units. Examination of normal and diabetic rat heart sections indicated that the latter contained abundant PAS-positive strands and nodules which corresponded to the distribution of anti type VI collagen reactivity. Moreover, immunoblots showed higher levels of Type VI collagen in diabetic than in normal heart extracts. Type VI collagen therefore appears to represent a major glycoprotein of myocardial extracellular matrix and to be implicated in diabetic cardiomyopathy.

Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle.

The potential role of degradative mechanisms in controlling the level of the dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 required for protein N-glycosylation has been explored in thyroid slices and endoplasmic reticulum (ER) vesicles, focusing on cleavage of the oligosaccharide from its lipid attachment and on the enzymatic removal of peripheral monosaccharide residues. Vesicle incubations demonstrated a substantial release of free Glc3Man9GlcNAc2 (at 30 min approximately 35% of that transferred to protein) which was inhibited in the presence of exogenous peptide acceptor and was sensitive to disruption of membrane integrity by detergent. In thyroid slices glucosylated oligosaccharides terminating in the di-N-acetylchitobiose sequence were also noted and these continued to be formed even during inhibition by puromycin of both protein synthesis and the attendant N-glycosylation. These observations indicated that the oligosaccharide originated from the lipid donor and suggested, together with previously reported similarities in substrate specificity and cofactor requirements, that the oligosaccharyltransferase can carry out in vivo both the hydrolytic and transfer functions. In addition to the release of the intact Glc3Man9GlcNAc2, we also obtained evidence that the lipid-linked oligosaccharide can be modified by the in vivo action of ER glycosidases. Since radiolabeling of the oligosaccharide-lipid in thyroid slices indicated a preferential turnover of the glucose residues, the possible existence of a glucosyltransferase-glucosidase shuttle was explored with the use of castanospermine. In the presence of this glucosidase inhibitor, the formation of under-glucosylated and nonglucosylated oligosaccharides was not observed, even under conditions of energy deprivation in which they accumulate. Glucosidase inhibition in ER vesicle incubations likewise prevented the appearance of incompletely glucosylated oligosaccharide-lipids. Studies employing the mannosidase inhibitor 1-deoxymannojirimycin in thyroid slices furthermore indicated that in vivo removal of at least one mannose residue from the dolichyl pyrophosphate-linked oligosaccharide can occur.

Biosynthesis of sulfated asparagine-linked complex carbohydrate units of calf thyroglobulin.

Thyroglobulin from colloid as well as from membrane fractions became radiolabeled upon incubation of calf thyroid slices with [35S]sulfate. The identity of the sulfate-labeled molecule was established by immunoprecipitation, polyacrylamide gel electrophoresis, Bio-Gel A-5m filtration, and DEAE-cellulose chromatography. Size analysis by gel filtration of [35S]glycopeptides and hydrazine-released oligosaccharides indicated that the sulfate was primarily located in the complex (unit B) carbohydrate units of thyroglobulin. Moreover, although [35S]sulfate-labeled oligosaccharides were cleaved by N-glycanase to the same extent as those labeled with [3H]mannose, they were not released by endo-beta-N-acetylglucosaminidase under conditions that led to the complete removal of polymannose carbohydrate (unit A). The failure of 35S-labeled glycopeptides and oligosaccharides to bind to immobilized Concanavalin-A indicated that the sulfate residues in calf thyroglobulin are located in carbohydrate units with three or more branches. No evidence for the occurrence of tyrosine sulfate was found upon examination of Pronase digests of radiolabeled thyroglobulin, and chemical analyses excluded the presence of this amino acid down to a level of 0.5 residues/polypeptide subunit. Studies with density gradient-separated membrane fractions as well as with puromycin indicated that sulfate addition is a late event in thyroglobulin biosynthesis which occurs in the Golgi compartment. Furthermore, it was observed that the nondimerized thyroglobulin subunit was much less sulfate labeled than the mature molecule. The location of the sulfated carbohydrate in a terminal portion of the calf thyroglobulin peptide chain was suggested by the observation that the subunit [mol wt (Mr) = 330,000] can undergo a transformation, presumably mediated by an endogenous protease, to a sulfate-free component (Mr = approximately 270,000) with the appearance of a 35S-labeled 60,000 Mr fragment; the release of a single sulfate-labeled peptide (Mr = 60,000) by mild trypsin treatment was consistent with a sequestration of sulfate groups in the thyroglobulin molecule.

Sulfate metabolism in the alloxan-diabetic rat: relationship of altered sulfate pools to proteoglycan sulfation in heart and other tissues.

The incorporation of [35S]sulfate into heart proteoglycans has been studied in normal and alloxan-diabetic rats by perfusion and in vivo administration of the isotope; in the latter situation, comparison was also made of radiolabeled sulfate utilization by several other tissues (kidney, liver, lung, muscle, testes and skin). The radiolabeled products were characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis and anion exchange chromatography, as well as by sizing of the glycosaminoglycan chains by gel filtration both before and after nitrous acid treatment. The most prominent band observed in the heart guanidine extract by electrophoresis had a molecular weight of 85,000 and minor components (Mr = 360,000 and 170,000) were also detected; approximately 20% of the proteoglycan associated [35S]sulfate was present in heparan sulfate chains. After perfusion the pattern, as well as the amount of radioactivity recovered from the diabetic heart, was similar to the normal heart. In contrast, after intraperitoneal injection of the [35S]sulfate, a substantial reduction in incorporation was found not only in heart but in several other tissues studied, although no qualitative differences were noted in the macromolecules formed by the two groups of animals. Measurement of the serum sulfate concentration indicated that the level in the alloxan-diabetic rat (1.23 mmol/l) was significantly less (p less than 0.01) than that of the normal rat (1.67 mmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)