Characterization of a 180 kDa molecule apparently reactive with recombinant L-selectin.
In the present study we identified a 180 kDa molecule (p180) in rat lymph nodes (LN) apparently reactive with silkworm derived recombinant L-selectin (LEC-IgG) in a Ca(2+)-dependent manner. Analysis of amino acid sequence revealed that p180 has a strong homology to the macrophage mannose receptor (MMR), which was corroborated by the observation that p180 reacted with polyclonal anti-alveolar MMR antibody and mannosyl-BSA-agarose. In agreement with this notion, the binding of p180 to the silkworm LEC-IgG was inhibited by alpha-methyl-D-mannoside. However, in sharp contrast to its reactivity against the silkworm LEC-IgG, p180 failed to bind LEC-IgG produced by COS-7 cells, suggesting that p180 reacted with the silkworm LEC-IgG through the recognition of oligomannose-type oligosaccharides expressed on the silkworm products and that the lectin activity of L-selectin was not involved in the interaction. These results, together with the immunohistochemical studies showing that p180 was absent from the majority of high endothelial venules (HEV) but present in medullary macrophages, led us to conclude that p180 obtained from LN lysates by the use of the silkworm LEC-IgG is not a physiological ligand for L-selectin, warning against the use of recombinant proteins expressed in the baculovirus/ silkworm expression system for the detection of carbohydrate ligands.
Kawashima
H
H
Department of Bioregulation, Osaka University Medical School, Suita, Japan.
Watanabe
N
N
Li
Y F
YF
Hirose
M
M
Miyasaka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D012831:Bombyx; D002846:Chromatography, Affinity; D019041:L-Selectin / Q000378:metabolism*; D037102:Lectins; D037181:Lectins, C-Type; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000378:metabolism*; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection
D000906:Antibodies; D037102:Lectins; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019041:L-Selectin