Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.
Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.
Rittershaus
C W
CW
Avant Immunotherapeutics, Inc., Needham, Massachusetts 02494-2725, USA.
Thomas
L J
LJ
Miller
D P
DP
Picard
M D
MD
Geoghegan-Barek
K M
KM
Scesney
S M
SM
Henry
L D
LD
Sen
A C
AC
Bertino
A M
AM
Hannig
G
G
Adari
H
H
Mealey
R A
RA
Gosselin
M L
ML
Couto
M
M
Hayman
E G
EG
Levin
J L
JL
Reinhold
V N
VN
Marsh
H C
HC
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002448:Cell Adhesion; D003167:Complement Activation / Q000187:drug effects*; D006224:Cricetinae; D004586:Electrophoresis / Q000379:methods; D005434:Flow Cytometry; D006023:Glycoproteins / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000032:analysis; D011485:Protein Binding; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D019087:Selectins / Q000378:metabolism*; D020298:U937 Cells; D015854:Up-Regulation
D006023:Glycoproteins; D009005:Monosaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D019087:Selectins