Human complement factor I glycosylation: Structural and functional characterisation of the N-linked oligosaccharides Stefanos A. Tsiftsoglou a, ⁎ , James N. Arnold b , Pietro Roversi c , Max D. Crispin b , Catherine Radcliffe b , Susan M. Lea c , Raymond A. Dwek b , Pauline M. Rudd b , Robert B. Sim a a MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England, UK b Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England, UK c Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England, UK Received 21 July 2006; received in revised form 7 September 2006; accepted 8 September 2006 Available online 16 September 2006 Abstract Factor I (fI) is a key serine protease that modulates the complement cascade by regulating the levels of C3 convertases. Human fI circulates in plasma as a heavily N-glycosylated (25–27% w/w) heterodimer composed of two disulphide linked chains, each carrying three N-linked oligosaccharide chains. It had been suggested that the oligosaccharides may have both structural and functional roles in the interactions with the natural substrate and the cofactor during a catalysis. The N-linked glycans of each fI chain were characterised in detail and the analysis revealed a similar composition of the glycan pools with both chains heavily sialylated. Disialylated structures were in excess over monosialylated ones: 55% over 40% for the heavy chain and 62% over 35% for the light chain. The dominant type of glycan identified on both chains was A 2 G 2 S 2 ,a biantennary structure with chains terminating in sialic acid linked to galactose. The glycan characterisation facilitated a strategy for the partial deglycosylation of the enzyme. Assessment of the proteolytic activities of the native and partially deglycosylated forms of fI showed that both forms of the enzyme have very similar proteolytic activities against C3(NH 3 ) indicating that the charged glycans of fI do not influence the fI- cofactor–substrate interactions. © 2006 Elsevier B.V. All rights reserved. Keywords: Human; Complement; Factor I; Serine protease; N-glycosylation; Sialylation Biochimica et Biophysica Acta 1764 (2006) 1757 – 1766 www.elsevier.com/locate/bbapap Abbreviations: 2-AB, 2-aminobenzamide; ABS, sialidase from Arthrobacter urefaciens for α(2–3) and α(2–6) linked sialic acid; Ac, N-acetyl; BKF, Fucosidase from bovine kidney; BTG, β-galactosidase from bovine testis; C3(H 2 O)/ C4(H 2 O), C3/C4 with hydrolysed thioester; C3(NH 3 ), A structural analog of C3(H 2 O). C3 (NH 3 ) is formed when the C3 thioester is cleaved by nucleophilic attack by NH 3 ; CV, Column Volume; fB, Complement factor B; fD, Complement factor D; fH, Complement factor H; fI, Complement factor I; FIMAC, Factor I Membrane Attack Complex; FPLC, Fast Performance Liquid Chromatography; GlcNAc, N- acetylglucosamine; GU, Glucose Unit; GuH, Recombinant β-N-acetylglucosaminidase from Streptococcus pneumonia; HC, Heavy chain of fI; HEPES, 2-[4-(2- hydroxyethyl-1-piperazine)]ethanesulphonic acid; iC3b, Inactivated C3b (cleaved by factor I); LC, Light chain of fI; LDLR-A, Low Density Lipoprotein Receptor type A; NanI, Recombinant sialidase from Streptococcus pneumonia preferentially cleaves α(2–3) linked sialic acid; N x , Asparagine with attached carbohydrate; NP- HPLC, Normal Phase High Performance Liquid Chromatography; PBS, Phosphate-Buffered Saline (Dulbecco A formula: 8.2 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 139 mM NaCl, 3 mM KCl, pH 7.4); PNGase F, Peptide-N 4 -(acetyl-beta-glucosaminyl)-asparagine amidase; SDS-PAGE sample buffer, 0.2 M Tris, 8 M urea, 2% (w/v) SDS, 2 mM EDTA, pH 8.0 with 0.001% (w/v) Bromophenol Blue and 40 mM 1,4-dithiothreitol (reducing conditions) or 20 mM iodoacetamide (alkylating conditions); SDS, Sodium Dodecyl Sulphate; SDS-PAGE, Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SP, Serine Protease; SRCR, Scavenger Receptor Cysteine Rich ⁎ Corresponding author. The CBR Institute for Biomedical Research, Harvard Medical School, 800 Huntington Avenue, Boston, MA 02115, USA. Tel.: +1 617 278 6661; fax: +1 617 278 6655. E-mail address: tsiftsoglou@cbr.med.harvard.edu (S.A. Tsiftsoglou). 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.09.007