Complex-type Carbohydrate Chains Research Articles

Deglycosylated mammalian β2-adrenergic receptors: Effect on radioligand binding and peptide mapping

Mammalian β-adrenergic receptors are glycoproteins containing both high-mannose and complex-type carbohydrate chains [G. L. Stiles, J. L. Benovic, M. G. Caron, and R. J. Lefkowitz (1984) J. Biol. Chem., 259, 8655–8663] . Endoglycosidase F treatment of β 2-adrenergic receptors results in the removal of at least two N-linked oligosaccharides, resulting in an increased mobility of the receptor peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( M r 62,000 to 49,000). In the present study the properties of the deglycosylated β 2-adrenergic receptor were assessed. Following deglycosylation, the β 2-adrenergic receptor recognized both agonists and antagonists with the same potency order and affinities as the glycosylated form. In addition, two-dimensional gel electrophoresis peptide mapping techniques applied to control and deglycosylated β 2-adrenergic receptors (both within and between species) demonstrated that there was a marked homology of the β 2-adrenergic receptor between species which are closely related phylogenetically. In addition, the glycan component of the receptor did not appear to interfere with the ability of proteinases to generate accurate peptide maps.

Structural changes of carbohydrate chains of human thyroglobulin accompanying malignant transformations of thyroid glands.

Carbohydrate moieties were released from human thyroglobulins prepared from normal and transformed, thyroid tissues by hydrazinolysis. The oligosaccharides thus prepared were fractionated by DEAE-cellulose, Bio-Gel P-4, concanavalin-A--Sepharose and Ricinus-communis-agglutinin--Sepharose columns and were characterized by exo-glycosidase and endo-glycosidase digestions, methylation analysis and 400-MHz 1H-NMR spectroscopy. These studies showed that the alterations accompanying malignancy occurred in complex-type carbohydrate chains and exhibited the following structural features: (a) a complex-type carbohydrate chain containing 1 phosphate in a diester linkage was present in all the malignant thyroglobulins. (b) Asialo-carbohydrate chains with biosynthetically incomplete structures were found in all of the malignant thyroglobulins. (c) Carbohydrate chains of higher molecular mass, which have repeating Gal-GlcNAc disaccharides and peripheral alpha-fucosyl residues were present in metastatic papillary carcinoma.

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains.

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Retinoic acid alters the proportion of high mannose to complex type oligosaccharides on fibronectin secreted by cultured chondrocytes.

Fibronectin synthesized by retinoic acid-treated chondrocytes exhibits a higher apparent subunit molecular weight in sodium dodecyl sulfate-polyacrylamide gels than fibronectin from untreated cultures. Analyses of peptide fragments show that retinoic acid treatment alters the apparent molecular weight of the collagen-binding fragment. Approximately one-sixth of the carbohydrate moieties on the collagen-binding fragment from control chondrocytes were sensitive to endo-beta-N-acetylglucosaminidase H, whereas the collagen-binding fragment from treated chondrocytes was more resistant to endo-beta-N-acetylglucosaminidase H. These findings indicate that chondrocyte fibronectin contains oligosaccharides of the high mannose or hybrid type in contrast to fibroblast fibronectin which only contains complex type carbohydrate chains and that retinoic acid treatment results in a higher proportion of complex type carbohydrate chains on chondrocyte fibronectin.

Comparison and partial characterization of guinea pig Ia alpha- and beta-chain oligosaccharides.

The structures of the N-linked oligosaccharides of mature guinea pig Ia molecules were partially characterized by serial lectin affinity analysis. Those Ia antigens that are thought to be allelic products (Ia.3,5 and Ia.4,5) were found to bear identical oligosaccharides, whereas differences in glycopeptide distribution were found for Ia antigens known to be products of separate I subregions (Ia.2 and Ia.4,5). The two predominant oligosaccharides present on alpha-chains from all three Ia molecules were of the high mannnose type and the triantennary or tetraantennary complex type. Two structurally distinct beta-chains were isolated from Ia.3,5 and Ia.4,5 molecules; beta 1 bore primarily triantennary or tetraantennary complex oligosaccharides, and beta 2 had predominantly biantennary complex-type carbohydrate chains. The composition and distribution of the oligosaccharide moieties of guinea pig Ia molecules indicate that there are structural features shared among guinea pig, murine, and human Ia antigens.

High-pressure liquid chromatography of isomeric oligosaccharides that form part of the complex-type carbohydrate chains of glycoproteins

High-pressure liquid chromatography of isomeric oligosaccharides that form part of the complex-type carbohydrate chains of glycoproteins

The primary structures of pancreatic ribonucleases from African porcupine and casiragua, two hystricomorph rodent species

The amino acid sequences of the pancreatic ribonucleases from African porcupine ( Hystrix cristata) and casiragua ( Proechimys guairae, a caviomorph rodent species related to the coypu) were determined. The ribonucleases were isolated form minces of pancreatic tissue which had been used for the extraction of the insulins. The results of the sequence determinations of residues 67–78 in both enzymes were ambiguous. Therefore, homology with other ribonucleases has been used in deriving these sequences. At position 94 aspartic acid was found, while ali other ribonuclease sequenced to date have asparagine at this position. This may indicate a specific deamidation as a result of the acidic conditions during the extraction of insulin. The amino acid sequence of African porcupine ribonuclease shows a close relationship with those of the South-American caviomorph rodents, which implies that the hystricomorph suborder of the rodents, to which both the African porcupine and the caviomorphs belong, is a natural (evolutionary) taxon. Both porcupine and casiragua ribonuclease are glycoproteins with complex-type carbohydrate chains attached to asparagine34.