ABSTRACT
A new mannose-recognizing lectin (MOL) was purified on an asialofetuin-column from fruiting bodies of Marasmius oreades grown in Japan. The lectin (MOA) from the fruiting bodies of the same fungi is well known to be a ribosome-inactivating type lectin that recognizes blood-group B sugar. However, in our preliminary investigation, MOA was not found in Japanese fruiting bodies of M. oreades, and instead, MOL was isolated. Gel filtration showed MOL is a homodimer noncovalently associated with two subunits of 13 kDa. The N-terminal sequence of MOL was blocked. The sequence of MOL was determined by cloning from cDNA and by protein sequencing of enzyme-digested peptides. The sequence shows mannose-binding motifs of bulb-type mannose-binding lectins from plants, and similarity to the sequences. Analyses of sugar-binding specificity by hemagglutination inhibition revealed the preference of MOL toward mannose and thyroglobulin, but asialofetuin was the strongest inhibitor of glycoproteins tested. Furthermore, glycan-array analysis showed that the specificity pattern of MOL was different from those of typical mannose-specific lectins. MOL preferred complex-type N-glycans rather than high-mannose N-glycans.
Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/genetics , Mannose-Binding Lectin/chemistry , Mannose-Binding Lectin/genetics , Marasmius/chemistry , Marasmius/genetics , Protein Multimerization , Amino Acid Motifs , Amino Acid Sequence , Animals , Asialoglycoproteins/chemistry , Asialoglycoproteins/metabolism , Cloning, Molecular , Fetuins/chemistry , Fetuins/metabolism , Fruiting Bodies, Fungal/chemistry , Fruiting Bodies, Fungal/genetics , Fruiting Bodies, Fungal/metabolism , Fungal Proteins/metabolism , Humans , Japan , Mannose-Binding Lectin/metabolism , Marasmius/metabolism , Molecular Sequence Data , Protein Binding , Rabbits , Thyroglobulin/chemistry , Thyroglobulin/metabolism , Trisaccharides/chemistry , Trisaccharides/metabolismABSTRACT
Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.
