Structural Characterization of Site-Specific N-Glycosylation of Recombinant Human Factor VIII by Reversed-Phase High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry

Abstract

The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subjected to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and the 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

“…Factor VIII contains a validated N-glycosylation site [22] that, in combination with other Nglycans, facilitates binding [23]. Moreover, N-glycosylation of DDR1 at a residue distal to the DSD (N211), that is conserved in DDR2, has been shown to affect receptor activation [24].…”

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

“…These endoglycosidases have been shown to cleave oligomannose bi and tri-antennary complex oligosaccharide structures from glycoproteins under native conditions (26,27). Complex tetraantennary structures that are found in low frequency (one of several potential sugars on only two residues the B domain of FVIII) are not cleaved by endoglycosidase Fs (5,6). The reactions were carried out at pH 6.5 or in the buffer provided by the manufacturer for 1 h at 37°C, if not stated otherwise.…”

“…The native full length protein has 25 potential N-glycosylation sites, including 19 sites in the B-domain and three each in light (two in A1, one in A2) and heavy (two in A3, one in C1) chains (4). Of these, 21 sites were identified as carrying oligosaccharides with 16-17 potential glycosylation sites occupied in the B-domain (5,6). In spite of the presence of one Asn-Xxx-Ser/Thr consensus sequence for N-linked glycosylation, the A2 domain of FVIII does not carry any oligosaccharides.…”

“…In spite of the presence of one Asn-Xxx-Ser/Thr consensus sequence for N-linked glycosylation, the A2 domain of FVIII does not carry any oligosaccharides. The A1 domain and the light chain carry two N-linked sugar chains each (4,5). FVIII binding to phosphatidylserine (PS)-containing platelet membrane surfaces is a critical step in the blood coagulation cascade and is necessary for the proper formation and anchoring of the tenase complex (7).…”

Galectin-1 and Galectin-3 Constitute Novel-Binding Partners for Factor VIII

“…11,12 The N-linked oligosaccharide chains of human plasma-derived and recombinant FVIII (rFVIII) have been characterized. 13,14 Bi-antennary, tri-antennary, and tetra-antennary complex-type chains, together with high-mannose-type sugars were identified. 13,15 Unusually, the complex N-linked oligosaccharides of plasma FVIII were also shown to express covalently linked ABO(H) blood group antigenic determinants.…”