N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.
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-beta-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) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-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 (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.
D000818:Animals; D030801:Animals, Genetically Modified; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003174:Complement C1 Inactivator Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000502:physiology; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003174:Complement C1 Inactivator Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins