Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding.
CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing alpha 2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Sia alpha 2-6Gal beta 1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A. (1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 microM) for Neu5Ac alpha 2-6Gal beta 1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the alpha 2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(alpha 2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.
Powell
L D
LD
Department of Medicine, University of California at San Diego, La Jolla 92093, USA.
Jain
R K
RK
Matta
K L
KL
Sabesan
S
S
Varki
A
A
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D037102:Lectins; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2; D013329:Structure-Activity Relationship
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; C497868:Cd22 protein, mouse; D015815:Cell Adhesion Molecules; D037102:Lectins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; C016735:sialooligosaccharides; D005340:Fibrinogen