IDENTIFICATION AND GENETIC-ANALYSIS OF A COMMON MOLECULAR VARIANT OF HISTIDINE-RICH GLYCOPROTEIN WITH A DIFFERENCE OF 2KD IN APPARENT MOLECULAR-WEIGHT

Two forms of histidine-rich glycoprotein (HRG) were detected on SDS-PA GE by silver staining and immunoblotting after isolation of the protei n from pooled plasma using immune-affinity chromatography followed by chromatography with heparin-Sepharose. Both forms were single-chain mo lecules and the apparent molecular weights of form 1 and form 2 were 7 7 kD and 75 kD respectively, Mendelian inheritance of both HRG forms w as observed in four families with 24 informative meioses, strongly sug gesting that the two forms are encoded by different alleles. The frequ ency of form 1 and form 2 in a group of 36 individuals was 0.35 and 0. 65 respectively. The difference between the two molecular variants was studied by direct sequence analysis of amplified exons of the HRG gen e from 6 individuals who were homozygous either for form 1 or form 2. Five amino acid polymorphisms in three different exons were observed: Ile/Thr in exon 4; Pro/Ser in exon 5; His/Arg, Arg/Cys and Asn/Ile in exon 7. Analysis of these polymorphisms in 20 volunteers showed that o nly the Pro/Ser polymorphism at position 186 in exon 5 was coupled to the form of the HRG protein. Ser was found in form 1 and Pro in form 2 . The presence of Ser at position 186 introduces a consensus sequence for a N-glycosylation site (Asn-X-Ser/Thr). By removing N-linked sugar s with N-glycanase, it could be demonstrated that the difference betwe en the two forms of HRG is caused by an extra carbohydrate group at As n 184 in form 1.