Characterization of a 180 kDa molecule apparently reactive with recombinant L-selectin
Authors: Kawashima, H.; Watanabe, N.; Li, Y-F.; Hirose, M.; Miyasaka, M.
Source: Glycoconjugate Journal, Volume 14, Number 3, 1 March 1997 , pp. 321-330(10)
Abstract: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 Ca2+-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 α-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. Abbreviations: BSA, bovine serum albumin; EDTA, ethylenediamine-N,N,N,N-tetraacetic acid; ELISA, enzyme-linked imunosorbent assay; HEV, high endothelial venule; IgG, immunoglobulin G; LN, lymph node; NP-40, Nonidet P-40; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PVR, poliovirus receptor; SDS, sodium dodecylsulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; sLeX, sialyl Lewis X; WGA, wheat germ agglutinin
Document Type: Regular Paper
Affiliations: Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, 2-2, Yamada-oka Suita 565 Japan
Publication date: 1997-03-01