Consensus repeat domains of E-selectin enhance ligand binding.
To study the structural characteristics of E-selectin necessary for mediating cell adhesion, we examined the role of the consensus repeat (CR) domains in E-selectin function. Soluble constructs containing different numbers of CR domains were stably expressed in Chinese hamster ovary cells, purified to homogeneity, and characterized. The minimum functional unit of soluble E-selectin consisted of the lectin (Lec) and epidermal growth factor (EGF) domains alone (Lec-EGF) as indicated by its ability to mediate in vitro HL-60 cell adhesion. However, E-selectin containing all six CR domains (Lec-EGF-CR6) at its COOH terminus was the most potent in blocking neutrophil or HL-60 cell adhesion to either immobilized E-selectin or cytokine-stimulated human umbilical vein endothelial cells. This increased potency of Lec-EGF-CR6 in blocking cell adhesion was not due to CR-mediated oligomerization of the protein. Lec-EGF-CR6 was most likely monomeric in solution, as judged by gel filtration fast protein liquid chromatography, membrane ultrafiltration, and chemical cross-linking analysis. Therefore, although the lectin and EGF domains are necessary and sufficient for mediating cell adhesion, the additional six CR domains, present in native E-selectin, contribute to the enhanced binding of E-selectin to its ligand.
Li
S H
SH
Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Burns
D K
DK
Rumberger
J M
JM
Presky
D H
DH
Wilkinson
V L
VL
Anostario
M
M
Wolitzky
B A
BA
Norton
C R
CR
Familletti
P C
PC
Kim
K J
KJ
D001483:Base Sequence; D001667:Binding, Competitive; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D019040:E-Selectin; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D015815:Cell Adhesion Molecules; D017931:DNA Primers; D019040:E-Selectin; D008024:Ligands; D011994:Recombinant Proteins