Characterization of N-linked oligosaccharides bearing sialyl lewis x moieties on an alternatively glycosylated form of soluble complement receptor type 1 (sCR1).
We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLe(x)) moiety. This modified glycoprotein could combine anti-complement activity with the ability to inhibit selectin-mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N-linked oligosaccharides in the alpha1-3 linkage, which is necessary for sLe(x) glycosylation. The glycoprotein purified from these cells was designated sCR1sLe(x), and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLe(x) was performed to confirm that the N-linked oligosaccharides possessed sLe(x) moieties and also to determine the extent of sLe(x) glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore-assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non-core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage-specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLe(x) moieties on the N-linked oligosaccharides of sCR1sLe(x). sCR1sLe(x) is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.
Picard
M D
MD
AVANT Immunotherapeutics, Inc., 119 Fourth Avenue, Needham, MA 02494, USA.
Pettey
C L
CL
Marsh
H C
HC
Thomas
L J
LJ
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016256:Lewis X Antigen / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D011951:Receptors, Complement / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D006023:Glycoproteins; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011951:Receptors, Complement; D011994:Recombinant Proteins; C082885:soluble complement inhibitor 1