Om P. Bahl

Dissociation and recombination of the subunits of human chorionic gonadotropin

Abstract Human chorionic gonadotropin has been dissociated into two subunits by urea. They have been separated by chromatography on DEAE-Sephadex. The subunits are non-identical as evidenced by electrophoresis, amino and carbohydrate composition and reassociate under suitable conditions.

A chemical method for the deglycosylation of proteins.

A simple and rapid chemical method for the deglycosylation of glycoproteins has been developed. The method involves the incubation of protein with trifluoromethanesulfonic acid at 0 degrees C from 0.5 to 2 h followed by the neutralization of the acid with aqueous pyridine at -20 degrees C. The method has been applied effectively to fetuin, ovine submaxillary mucin, ovine lutropin, and human choriogonadotropin. In 1 h almost all of N- and O-linked carbohydrates from ovine lutropin and human choriogonadotropin, with the exception of the linkage N-acetylglucosamine or N-acetylgalactosamine, were removed. Similarly, in 1 h all N-linked carbohydrates, excepting again the linkage sugar, in fetuin were degraded. Longer reaction times up to 2 h completely removed the O-linked carbohydrate chains from fetuin and ovine submaxillary mucin. The deglycosylated hormones thus prepared retained their immunological and biological activities.

[28] Enzymatic deglycosylation of glycoproteins

Publisher Summary Deglycosylation of glycoproteins is important in elucidation of structure, function, and biosynthesis of biologically significant glycoproteins. Enzymatic deglycosylation can be brought about by exoglycosidases either used sequentially or in a mixture or by endoglycosidases. The chapter discusses the conditions and the processes of deglycosylation of glycoproteins by exoglycosidases and by endoglycosidases. Denatured glycoproteins are more susceptible to hydrolysis with these enzymes than glycoproteins in their native conformation. Complete deglycosylation of glycoproteins in their native conformation can, however, be achieved by using much higher concentrations of the enzyme. The precise conditions for hydrolysis including enzyme concentration and the incubation time vary with the substrate and, therefore, should be determined for each unknown glycoprotein separately. Despite the discovery of several endoglycosidases that have facilitated the investigations on the structure and biosynthesis of carbohydrates, there still remains a paucity of enzymes with broad specificity that can cleave carbohydrates from glycoproteins in their native forms. There is still a need to continue the efforts to search for new endoenzymes.

[27] Chemical deglycosylation of glycoproteins

Publisher Summary Deglycosylation of glycoproteins is necessary for a number of studies such as in the structural determination of polypeptide chains, establishment of structure and function relationships of carbohydrates, 1-5 and their biosynthesis in glycoproteins. In all these studies it is important that the conditions used for chemical deglycosylation do not cause an impairment of the physicochemical integrity of the polypeptide chain. Two chemical reagents in the anhydrous form, trifluoromethanesulfonic acid (TFMS) and hydrogen fluoride (HF), have been employed for the deglycosylation of glycoproteins. TFMS by the modified procedure described herein is definitely superior to HF. It is much more potent, sensitive, convenient to use than HF because it does not require any special handling. To prevent any secondary reactions such as the alkylation of the polypeptide chains during deprotection of the protecting group, the use of anisole was made as a scavenger. This chapter discusses the chemical deglycosylation procedure by TFMS and HF in detail.

Human chorionic gonadotropin: Amino acid sequence of the α and β subunits

Summary The linear amino acid sequences of the α and β subunits of human chorionic gonadotropin have been determined. HCG-α has 92 and HCG-β 139 amino acid residues. There is more homology between HCG-α and ovine LH-α than between HCG-β and ovine LH-β.

Effect of deglycosylation on the subunit interactions and receptor binding activity of human chorionic gonadotropin.

Summary The role of carbohydrate in the subunit interactions and in the binding to the receptor was assessed by the deglycosylation of the individual α- and β-subunits of hCG with trifluoromethane sulfonic acid (TFMS) at 0° for 5 h. The treatment with TFMS removed about 90% and 81% of the carbohydrates from hCG-α and hCG-β subunits respectively, without affecting the protein core as evidenced by their amino acid analyses, immunological activities and molecular weights, determined by SDS-gel electrophoresis. The deglycosylated subunits not only were able to reassociate completely as shown by the amino acid composition of the reconstituted deglycosylated hCG, but also recovered their immunological and receptor binding properties (90–105%) comparable to the native hCG. The above data strongly suggest that the carbohydrates in both subunits are not required in the interaction of the subunits and in the receptor binding activity of the hormone.

The role of carbohydrate in human choriogonadotropin (hCG) action. Effects of N-linked carbohydrate chains from hCG and other glycoproteins on hormonal activity☆

Deglycosylation of gonadotropins and thyrotropin results in a major loss of hormonal bioactivity, while not impairing receptor-binding activity. However, a direct role of the glycan moieties in hormonal signal transduction has not been demonstrated. The addition of carbohydrate chains together with the deglycosylated hormone does not restore the hormonal activity. In contrast, glycopeptides were found to inhibit human choriogonadotropin (hCG)-stimulated adenylyl cyclase activity and hCG binding to its receptor. An inhibition of hCG-stimulated adenylyl cyclase activity but not hCG binding to receptor by glycopeptides specifically from hCG, has previously been reported as a lectin-like membrane component has been implicated in hCG action. In the present study we have shown that glycopeptides and oligosaccharides prepared from hCG, transferrin, fetuin, alpha 1-acid glycoprotein and ovalbumin inhibit the binding of hCG to its receptor. The inhibition was also observed with a highly purified preparation of the receptor, thus suggesting a lack of involvement of other lectin-like membrane components as previously proposed. We suggest that a lectin-like interaction with the hormone, if any, involves the receptor itself. Adenylyl cyclase activity stimulated by hCG, isoproterenol or forskolin was inhibited by oligosaccharides, indicating a non-specific interaction. Our results suggest that Asn-linked oligosaccharide chains from various glycoproteins perturb hCG-receptor interactions through a putative carbohydrate binding site on the receptor.

Role of carbohydrate in human chorionic gonadotropin: Deglycosylation uncouples hormone-receptor complex and adenylate cyclase system

Abstract Previous work has shown that deglycosylation of human chorionic gonadotropin (hCG) does not affect its receptor binding characteristics, but its ability to stimulate intracellular cyclic AMP accumulation and steroidogenesis in ovarian cells is abolished. To identify the site at which carbohydrate of hCG is involved in the mechanism of action of the hormone, we have studied adenylate cyclase activity in ovarian membrane preparations in response to deglycosylated and native hCG. The deglycosylated hCG does not stimulate adenylate cyclase of ovarian membrane preparation and also it acts as an inhibitor of hCG action. Data are presented to show that both hCG- and catecholamine receptors are coupled to the same adenylate cyclase complex. Since adenylate cyclase activity in the presence of deglycosylated hCG remains still responsive maximally to catecholamines, it indicates that the adenylate cyclase complex is functional and is unaffected by the interaction of deglycosylated hCG to its receptor. This is further supported by the fact that the deglycosylated hCG does not impair the maximal stimulation of adenylate cyclase by guanine nucleotides. Thus, the site of action of the carbohydrate of hCG is prior to the coupling of the hormone-receptor complex and the adenylate cyclase system.

Glycosidases. Ligands for affinity chromatography: I. Syntheses of 1,2-transp-aminophenyl 1-thio-D-glycopyranosides

Abstract 1,2-transp-Aminophenyl 1-thioglycosides derived from β- D -galactose, β- D -fucose, and α- D -mannose were synthesized as potential ligands for the purification of β- D -galactosidase and α- D -mannosidase by affinity chromatography. The appropriate acetylglycosyl bromides were condensed with p-nitrothiophenol in the presence of potassium hydroxide. Deacylation of the 1,2-transp-nitrophenyl O-acetyl-1-thio- D -glycopyranosides thus obtained, followed by reduction with hydrogen over palladium on barium sulfate, afforded the desired p-aminophenyl 1-thioglycopyranosides.

Polyclonal antibodies against the polypeptide and carbohydrate epitopes of recombinant human choriogonadotropin β-subunit ☆

In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin beta subunit (hCG beta) using the baculovirus-insect cell expression system. The rhCG beta was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCG beta was similar to hCG beta in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG beta was produced. The antiserum was found to be almost identical to anti-hCG beta in binding to hCG beta as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using 125I-hCG beta as a tracer. Further characterization of the anti-rhCG beta antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCG beta antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG beta, rhCG and thyroglobulin and failed to react with transferrin, alpha 1-acid glycoprotein and hCG alpha, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCG beta also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCG beta since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG beta-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCG beta and not in hCG beta. The eluate from the hCG beta-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCG beta and hCG beta. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the intracellular processing of the N-linked oligosaccharides.(ABSTRACT TRUNCATED AT 400 WORDS)