The adhesive properties of recombinant soluble L-selectin are modulated by its glycosylation.
The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocyte's ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin-ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the alpha 2-3 position, while alpha 2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.
Fieger
C B
CB
Institut für Klinische Chemie und Pathobiochemie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Germany.
Emig-Vollmer
S
S
Petri
T
T
Gräfe
M
M
Gohlke
M
M
Debus
N
N
Semmler
W
W
Tauber
R
R
Volz
B
B
D000269:Adhesives / Q000737:chemistry*; D000581:Amidohydrolases; D002448:Cell Adhesion; D002460:Cell Line; D004730:Endothelium, Vascular / Q000737:chemistry*; D005434:Flow Cytometry; D006031:Glycosylation; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D019041:L-Selectin / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008024:Ligands; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D014162:Transfection
D000269:Adhesives; D008024:Ligands; D011994:Recombinant Proteins; D019041:L-Selectin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase