Structural Analysis of Bikunin Glycosaminoglycan

Abstract

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein.Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

Sulfation of the Bikunin Chondroitin Sulfate Chain Determines Heavy Chain·Hyaluronan Complex Formation

“…Urine fraction 2 was found to contain un-, 4-, 6-, and disulfated CS disaccharides, whereas urine fraction 1 was found to contain un-and 4-sulfated CS disaccharides. The CS composition of purified I␣I from human plasma has been reported to contain un-and 4-sulfated disaccharides in a ratio of 3:1 (26,27). This is in agreement with fraction 1 analyzed in this study that was found to be the major population of bikunin in each of the fluids analyzed.…”

“…The CS chains of urinary bikunin and plasma I␣I have been found to contain 15 Ϯ 3 disaccharides of which ϳ5 disaccharides are 4-sulfated on GalNAc and clustered near the reducing end (23)(24)(25)(26)(27). The CS chain linkage region of bikunin in urine and plasma has been shown to contain a disulfated hexasaccharide (24,25) on its non-reducing end, whereas other groups have suggested that the non-reducing end is monosulfated on the GalNAc residue (9,28).…”

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

“…The two HCs are positioned in close proximity, with HC2 toward the reducing end of the CS chain (8). The CS chain is 12-18 disaccharides long, and on average, every fourth disaccharide is sulfated (C-4-S) (8,9). The remaining disaccharides are unsulfated (C-0-S) (8,9).…”

The Transfer of Heavy Chains from Bikunin Proteins to Hyaluronan Requires Both TSG-6 and HC2

“…The bikunin proteins inter-␣-inhibitor (I␣I), pre-␣-inhibitor (P␣I), and HC2⅐bikunin contain the serine proteinase inhibitor bikunin, which is a member of the I2 Kunitz-BPTI inhibitor family (MEROPS data base) (5,6). Bikunin carries a single chondroitin sulfate (CS) chain originating from Ser-10 (7) composed of both unsulfated chondroitin and chondroitin 4-sulfate (8,9). Furthermore the bikunin proteins contain different heavy chains (HCs) selected from a group of five proteins (10), but only three of the five homologous HCs have been identified in complex with bikunin.…”