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Glycobiology Advance Access originally published online on September 26, 2003
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Glycobiology vol 14 no 1 pp. 51-64, 2004
© Oxford University Press 2004; all rights reserved.

N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits

Kate Koles1,4, Patrick H. C. van Berkel2,5, Frank R. Pieper5, Jan H. Nuijens5, Maurice L.M. Mannesse5, Johannes F.G. Vliegenthart4 and Johannis P. Kamerling3,4

4 Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands, and 5 Pharming Technologies BV, Archimedesweg 4, P.O. Box 451, NL-2300 AL Leiden, The Netherlands

Received on July 22, 2003; revised on September 10, 2003; accepted on September 11, 2003

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) ({alpha}2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were ({alpha}1-6)-core-fucosylated or ({alpha}1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual ({alpha}2-3)- and ({alpha}2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.

1 Present address: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128

2 Present address: GenMab BV, Jenalaan 18d, NL-3584 CK Utrecht, The Netherlands

3 To whom correspondence should be addressed; e-mail: j.p.kamerling{at}chem.uu.nl


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