Changes In Protein Glycosylation Research Articles

Heat-shock induced protein modifications and modulation of enzyme activities.

Upon heat stress, the cell physiology is profoundly altered. The extent of the alterations depends on the severity of the stress and may lead to cell death. The heat shock response is an array of metabolic changes characterized by the impairment of major cellular functions and by an adaptative reprogramming of the cell metabolism. The enhanced synthesis of the HSPs is a spectacular manifestation of this reprogramming. Numerous post translational modifications of proteins occur in response to heat stress and can be related to altered cellular functions. Some proteins are heat-denatured and temporarily inactivated. Heat-denaturation is reversible, chaperones may contribute to the repair. The extent of heat-denaturation depends on the cell metabolism: (a) it is attenuated in thermotolerant cells or in cells overexpressing the appropriate chaperones (b) it is enhanced in energy-deprived cells. Covalent modifications may also rapidly alter protein function. Changes in protein glycosylation, methylation, acetylation, farnesylation, ubiquitination have been found to occur during stress. But protein phosphorylation is the most studied modification. Several protein kinase cascades are activated, among which the various mitogen activated protein kinase (MAP kinase) cascades which are also triggered by a wide range of stimuli. As a possible consequence, stress modifies the phosphorylation status and the activity of components from the transcriptional and translational apparatuses. The same kinases also target key enzymes of the cellular metabolism. Protein denaturation results in constitutive hsp titration, this titration is a signal to trigger the heat-shock gene transcription and to activate some of the protein kinase cascades.

Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

Proteins of crustacean exoskeleton: III. Glycoproteins in the Bermuda land crab Gecarcinus lateralis

AbstractChitin and protein are major components of crustacean exoskeletons. In insect cuticles, glycosylation is one of the predominant post‐translational modifications of the proteins. Investigations of glycosylation of crustacean exoskeletal proteins are much more limited. We have used lectins to follow changes in glycosylation of proteins in the exoskeletal layers of the Bermuda land crab Gecarcinus lateralis at selected stages of the intermolt cycle. Proteins extracted from the individual layers during anecdysis and late proecdysis as well as from the layers of exuviae were electrophoresed on sodium dodecyl sulfate (SDS)‐polyacrylamidegels, blotted to polyvinylidene difluoride membranes, and reacted with biotinylated lectins. Seven lectins were tested: concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ulex europaeus agglutinin(UEA I), Ricinus communis agglutinin (RCA120), and peanut agglutinin (PNA). Con A, which binds primarily to mannose residues, produced the strongest signals. Duringanecdysis, most of the binding occurred with protein bands larger than 31 kDa, while in lateproecdysis and in layers from exuviae, small as well as large proteins were bound. N‐glycosidase F digestion of electrotransferred proteins and subsequent treatment with Con A indicated both N‐linked and O‐linked glycosylation. SBA, which binds to both α‐and β‐linked N‐acetylgalactosamine, was the second most reactive lectin, and the number of exoskeletal proteins to which it bound also increased in late proecdysis. Little or no binding of DBA, RCA120, or PNA, which react with α‐linked N‐acetylgalactosamine, indicated that the N‐acetylgalactosamine was β‐linked. The specificity of binding of Con A or SBA was demonstrated by inhibition with methyl‐α‐D‐mannopyranoside and N‐acetyl‐D‐galactosamine, respectively. Binding of UEAI revealed the presence of fucose residues on a few proteins. As the exoskeleton was degraded during proecdysis, protein bands that were not detected by lectin binding during anecdysis were modified in such a way that they became accessible to lectins. © 1995 Wiley‐Liss, Inc.

Effect of dietary fiber at weaning on protein glycosylation in the rat small intestine

Changes in protein glycosylation which can be modulated by dietary factors are observed in the rat intestinal mucosa at the weaning period. Experiments were performed to evaluate the involvement of dietary fibers in the regulation of such modifications. Groups of rats were abruptly weaned at 19 days of age on semi-synthetic diets differing in dietary fiber content (fiber-free, 10% pectin or 10% cellulose) given for 4 and 10 days. Glycoprotein sugars, activities of the fucosylation pathway and caecal contents were analyzed. Neutral sugar contents in glycoproteins of the small intestinal mucosa were increased in the fiber-fed groups as compared to fiber-free group, only after 4 days but not after 10 days of diet. Diet-induced modifications in the glycoprotein fucose content of the small intestinal mucosa are partly explained by the coordinated evolution of different activities involved in the fucosylation pathway (GDP-fucose production and breakdown, fucosyltransferase and fucosyltransferase inhibitor). Caecal contents of short chain fatty acids were significantly different between the three groups after 4 but not after 10 days of diet. There was no correlation between caecal short chain fatty acid contents and activities involved in the fucosylation pathway. The introduction of dietary fibers at weaning induced marked but transient changes in glycoprotein sugars and the fucosylation pathway. The results demonstrate that fucosylation is regulated in several ways including changes in fucosyltransferase activity but that caecal fermentation of dietary fibers was not directly responsible for the observed changes.

A general method for the complete deglycosylation of a wide variety of serum glycoproteins using peptide-N-glycosidase-F

Many indirect serum studies show changes in protein glycosylation in disease, but further progress will require direct investigation of oligosaccharide composition. Current methods of deglycosylation using PNGase-F often result in incomplete removal of oligosaccharides. This is an unsatisfactory situation because only small quantities of material are often available in clinical studies, glycosylation changes may occur in only a small proportion of the molecules and quantification of the released oligosaccharides may be unreliable. The ability of PNGase-F to deglycosylate haptoglobin (Hp) under different conditions has been investigated. Oligosaccharides were completely removed from 50μg of Hp by treatment for 24 h with PNGase-F in 50 mmol/l ammonium formate buffer, pH 8.6, in combination with sodium dodecyl sulphate, mercaptoethanol and Nonidet P40. This modified procedure was equally effective at removing oligosaccharides from other serum glycoproteins (α1 glycoprotein, α1, transferrin) and fetuin, and its efficiency was independent of the polymeric structure of the molecule or the amount of glycosylation. The method has the additional advantage of using 20% less enzyme than previous methods, which substantially reduces costs.

Analysis of Glycoproteins Separated by Two-Dimensional Gel Electrophoresis Using Lectin Blotting Revealed by Chemiluminescence

The carbohydrate structures of blotted glycoproteins can be analyzed by probing with lectins. The objective of the present work was to optimize the lectin blotting of human plasma glycoproteins separated by two-dimensional gel electrophoresis and the detection by the sensitive chemiluminescence method. The proposed detection method was found to be ten times more sensitive than a standard colorimetric reaction. Furthermore, the generated signals are detected on a X-ray film and provide a permanent record. The method is also very reliable when compared to the colorimetric detection. The present procedure for glycoprotein analysis is particularly well suited for screening changes in glycosylation of proteins in biological samples.

Protein glycosylation changes in Alzheimer's disease

Protein glycosylation changes in Alzheimer's disease

Induction of glucose regulated protein (grp78) and inducible heat shock protein (hsp70) mRNAs in rat brain after kainic acid seizures and focal ischemia.

Specific probes were obtained using PCR cloning from rat brain for the 78 kDa glucose regulated (grp78), inducible 72 kDa (hsp70) as well as constitutive 73 kDa (hsc73) heat shock mRNAs. Grp78 and hsc73 were expressed in normal rat brain whereas hsp70 was not. Subcutaneous injection kainic acid (10 mg/kg) produced seizures and induced all three mRNAs. The induction of grp78 and hsp70 mRNAs occurred within 2 h, peaked between 6-8 h, persisted for 48 h, and returned to control levels by 72 h. Expression of the grp78 and hsp70 mRNAs after focal ischemia progressively increased with occlusion durations from 15-120 min in the cerebral cortex. Though grp78 and hsp70 mRNAs were induced modestly in the striatum by 15 min of ischemia, longer durations of ischemia were characterized by little change in the grp78 mRNA levels and relatively lower levels of hsp70 expression. This result indicates that progressive increases in the duration of ischemia in brain, prior to infarction, may produce proportional increases in transcription of the heat shock genes. However, once the duration of ischemia is long enough to produce infarction, this severely limits the availability of ATP which blocks transcription of the heat shock genes. In conclusion, concurrent induction of the heat shock genes suggests that kainic acid seizures and focal ischemia induce several different stress responses in brain cells caused by denaturation of proteins, changes of protein synthesis, and changes of protein glycosylation.

Regulatory role of GM3 ganglioside in alpha 5 beta 1 integrin receptor for fibronectin-mediated adhesion of FUA169 cells.

Mouse mammary carcinoma mutant cell line FUA169, characterized by high GM3 ganglioside content, was established from parent cell line FM3A/F28-7, which has high lactosyl ceramide (LacCer) content but no GM3. FUA169 displays no changes in protein glycosylation, and is a typical glycolipid mutant differing from its parent in that it contains high quantities of GM3 and GlcCer, but no LacCer (see accompanying paper; Tsuruoka, T., Tsuji, T., Nojiri, H., Holmes, E. H., Hakomori, S. (1993) J. Biol. Chem. 268, 2211-2216). In contrast to parent F28-7 cells, FUA169 cells showed clear adhesion to fibronectin (FN). Several lines of evidence indicate that adhesion of FUA169 cells to FN requires the presence of GM3, which supports the function of integrin receptor. (i) Both FUA169 and F28-7 cells express the same quantity of FN integrin receptor, which consists of alpha 5 beta 1 (sensitive to RGDS peptide) and alpha 4 beta 1 (sensitive to CS1 peptide). However, adhesion to FN-coated plates, regardless of type of FN, was much higher for FUA169 than for F28-7 cells. (ii) F28-7 cells, which normally lack GM3 and adhere only weakly to FN, acquired GM3 during incubation in GM3-containing medium, and subsequently adhered strongly to FN. (iii) Cholesterol-lecithin liposomes (cholesterol was 14C-labeled) incorporating alpha 5 beta 1 receptor isolated from human placenta showed clear adhesion to FN-coated plates, and this adhesion was completely inhibited by RGDS peptide and by anti-beta 1 mAb ZH1. When liposomes included a moderate quantity of GM3 (0.22-0.44 micrograms (0.2-0.4 nmol)/55 micrograms of phosphatidylcholine, 33 micrograms of cholesterol, 5 micrograms of alpha 5 beta 1 in liposome), adhesion was enhanced significantly. In contrast, adhesion was greatly reduced below control level for alpha 5 beta 1 liposomes containing a higher quantity (2.2 micrograms; > 2 nM) of GM3. Adhesion to FN was also inhibited, but never enhanced, for alpha 5 beta 1 liposomes with similar composition but containing 0.4 nmol (or other quantities) of LacCer or GlcCer instead of GM3. These findings suggest that the greater adhesion to FN by FUA169 cells, relative to parent F28-7 cells, is due to functional support by GM3 of alpha 5 beta 1 integrin receptor.

Comparison of sialyl- and α-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased β-galactoside α-2,6-sialyltransferase

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with α-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha- ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared α-1,3-galactosyltransferase, α-2,3- and α-2,6-sialytransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of α-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of β-galactoside α-2,6-sialyltransferase in ras-transfectant although α-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of α-2,6-sialyltransferase.

Cell surface oligosaccharides on Dictyostelium during development

ABSTRACT Developmental studies of the changes in protein glycosylation are useful in elucidating the role of oligosaccharides in biological events. We have used the chemical technique, hydrazinolysis, to release oligosaccharides from cell surface glycoproteins of Dictyostelium discoideum. Oligomannose type, xylose- and fucose-containing oligosaccharides were found to be present. The charged oligosaccharides contained sulphate and mannose 6-phosphate residues; no sialic acid was detected. The charged oligosaccharides also contained significant amounts of xylose, arabinose, fucose and galactose, as well as mannose and N-acetylglucosamine, which were the main constituents of the neutral glycans. By monitoring the chemical characteristics of the liberated oligosaccharides, dramatic changes in both the charge and size distribution of cell surface oligosaccharides were observed throughout the 24 h period of cell development. A comparison, however, between the neutral glycan structures of prestalk and prespore cells, over the same time frame showed no dramatic differences. Discoidin, a lectin present on the cell surface of 8 h cells, was found not to be glycosylated. Affinity chromatography using immobilised discoidin was used to probe a sugar library made from the cell surface glycoproteins of 8h cells. Discoidin was found to bind selectively an oligosaccharide with the structure Manα3(Manα6)(Xylβ2)Manβ4GlcNAc. This oligosaccharide lacks a conventional N,N′-diacetyl- chitobiose core and has only been previously observed in plant glycoproteins. Peptide-N-glycosidase F treatment of horseradish peroxidase released an identical structure, confirming that the oligosaccharide was not a degradation fragment of the hydrazine. The oligosaccharide was found to inhibit discoidin-mediated haemagglutination with a Ki of 0.75 mM, a concentration approximately 100 times lower than that for galactose. The correlation between changes in the amoebal plasma membrane oligosaccharide structures and the biological events occurring at different stages of development such as cell-cell adhesion and cell-substratum attachment suggest an important role for sugars in these processes.

Changes in protein glycosylation during chick embryo development

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to- N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo- β- N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.