Abstract
beta-Glucan receptors are present on mammalian leukocytes and initiate phagocytosis of particulate yeast beta-glucans, such as zymosan particles. Human monocytes and U937 cells express two membrane proteins of 180 and 160 kDa, each of which binds particulate yeast glucan through a 20-kDa polypeptide constituent. In this report, the structural composition of the two beta-glucan receptors and the biochemical properties of their polypeptide constituents were examined. The 180-kDa receptor was composed of three disulfide-linked polypeptides of 95, 60, and 20 kDa, whereas the 160-kDa receptor was a multimer of two polypeptides of 27 and 20 kDa. Unlike other receptor constituents, the 20-kDa polypeptide was nonglycosylated and focused at two distinct isoelectric points. Immunoblots of the focused polypeptides showed the two 20-kDa variants and the 95-kDa subunit to be constitutively tyrosine-phosphorylated, a feature not previously reported for receptors on human mononuclear phagocytes. Dephosphorylation of the receptor proteins resulted in the loss of antigenic phosphotyrosine without affecting the antigenicity of either 20-kDa variant for the anti-idiotypic antibody to beta-glucan receptors. Separate analysis of the 160-kDa receptor showed it contained both variants of the 20-kDa polypeptide. Thus, the 20-kDa subunit constituent of the two beta-glucan receptors is a functionally and chemically unique polypeptide with apparent microheterogeneity in its primary structure.
Highlights
We present the structural composition of the 180- and 160-kDa J3-glucan receptor proteins on U937 cells and characterize the polypeptide subunit chains of the two multimeric proteins
To determine whether the receptors were homogeneous in their content of 20-kDa polypeptide variants, the 160-kDa receptor was purified by immunoadsorption with mAb ACIA12 (Fig. 2) from a preparation of receptors isolated with the anti-Id
The present studies demonstrate the structural composition of two 13-glucan receptors on human myelomonocytic U937 cells and investigate the biochemical properties of their disulfidelinked subunit constituents
Summary
Materials-Acrylamide, SDS, glycine, a mpholytes, nitrocellulose, and all other electrophoretic supplies were obtained from Bio-Rad; cystatin from Aldrich ; pepstatin, leupeptin, phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, Nonidet P-40, sodium thioglycollate, periodic acid solution, Schiff's reagen t , and general chemicals from Sigma; and the human myelomonocytic U937 cell line was from the American Type Culture Collection (Rockville, MD). For carbohydrate analysis by periodic acid-Scruff (PAS) reagent, reduced samples of purified receptor protein were separated by SDSPAGE, electroblotted onto nitrocellulose, treated for 15 min with 1% periodic acid in 3% acetic acid, and washed with several changes of distilled water. The oxidized samples were stained for 15 min in the dark with fresh Schiff's reagent, treated for 5 min with 0.5% sodi um m-bisulfite, washed, and stored in the dark By this procedure, the carbohydrate in ovalbumin (4%) and rabbit IgG (3%) was detectable in blots of 0.1 J.Lg of electrophoresed protein. Amino Acid Composition-Receptor proteins in reduced samples were resolved by SDS-PAGE, transferred to lmmobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) in 0.01 M 3-cyclohexylamino-1-propanesulfonic acid , pH 11.25, containing 10% methanol, stained briefly with Coomassie Blue, and destained. Analysis was carried out for 1.5, 3.8, 16.6, and 23.2 pmol of the 95-, 60-, 27-, and 20-kDa polypeptides, respectively
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