Molecular heterogeneity of the β-core fragment of human chorionic gonadotrophin

Abstract

We have analysed the structure and composition of the beta-core fragment of human chorionic gonadotrophin (beta C-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced beta C-hCG demonstrated two major bands with apparent molecular weights of M(r) 8900 and M(r) 7500. The molecular weight of the agalacto beta C-hCG was estimated to be M(r) 10,218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported beta 6-40 (M(r) 5000) and beta 55-92 (M(r) 5300) peptides of the beta hCG subunit. The results showed that 56-78% of beta C-hCG molecules of molecular weight M(r) 12,800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2.8, glucosamine 5.3, galactosamine 0.3, fucose 1.7 and sialic acid 3.0. Approximately 22-44% of the beta C-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately M(r) 10,000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of beta C-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues.

Screening Method to Evaluate Point-of-Care Human Chorionic Gonadotropin (hCG) Devices for Susceptibility to the Hook Effect by hCG β Core Fragment: Evaluation of 11 Devices

“…We observed a major peak with molecular weight of approximately 9.5 KDa. The MALDI/TOF result was consistent with reduced purified hCG␤cf with molecular weights of approximately 7.5 and 8.9 KDa observed on SDS-PAGE by de Medeiros et al (10 ) (see Supplemental Fig. 1, which accompanies the online version of this article at http://www.clinchem.org/ content/vol60/issue4).…”

“…A single band with a molecular weight of approximately 17 KDa was observed. The SDS-PAGE result was comparable to that of nonreduced hCG␤cf purified with the Sephadex G-75 column, as previously reported (10 ). We also assessed purity by MALDI/TOF mass spectrometry analysis.…”

Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance

“…lower MAL-II binding). The presence in urine of substantial amounts of hCGβcf, which contains minimal , de Medeiros et al 1993 or no , Jacoby et al 2000 galactose, may explain the overall lower levels of RCA-I binding for hCG from patient urine specimens than from choriocarcinoma cell culture supernatant (36 RU RCA-I per 150 RU immunoreactive urinary hCG compared to 180 RU RCA-I per 200 RU immunoreactive cellular hCG (Kelly et al 2005).…”

“…Both binding modes of GNA could be exploited on the abbreviated carbohydrates of hCGβcf. Although many hCGβcf oligosaccharides are highly truncated, not all of the glycoforms contain terminal mannose (de Medeiros et al 1993). Almost 20% contain GlcNAc moieties distal to the mannose core (Jacoby et al 2000), and these were postulated to derive from the low percentage of choriocarcinoma-like bi-and triantennary hCG glycoforms found in normal pregnancy.…”

Formas moleculares da gonadotrofina coriônica humana: características, ensaios e uso clínico

“…Seu núcleo protéico contém 73 aminoácidos estruturados em duas pequenas cadeias polipeptídicas, resíduos 6 a 40 e 55 a 92 da βhCG, ligadas por cinco pontes dissulfídicas não covalentes. Sua estrutura glicídica contém duas ramificações de oligossacarídeos complexos N-ligados fixos aos peptídeos Asn-13 e Asn-30 59,60 . Estas ramificações de carboidratos são também degradadas, contendo cada uma apenas 5 a 11 resíduos de açúcar 60 .…”