Recombinant soluble human CD69 dimer produced in Escherichia coli: reevaluation of saccharide binding.
We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan.
Childs
R A
RA
Imperial College School of Medicine, Northwick Park Campus, Harrow, Middlesex, HA1 3UJ, United Kingdom.
Galustian
C
C
Lawson
A M
AM
Dougan
G
G
Benwell
K
K
Frankel
G
G
Feizi
T
T
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D004220:Disulfides; D000939:Epitopes; D037181:Lectins, C-Type; D009005:Monosaccharides; D010446:Peptide Fragments; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D000099192:CD69 Antigens; C007789:fucoidan