Exploitable results
The biochemical analysis of the glycoprotein isolated from cultured embryonic cells of Drosophila melanogaster included the examination of its amino acid and sugar composition. A high proportion of Ser, Thr and Pro, constituting more than 30% of the total number of amino acids was revealed. This is typical for vertebrate O-glycosylated (mucin-type) glycoproteins. Monosaccharide composition revealed by lectin studies and chemical analysis was quite typical for vertebrate O-glycoproteins with GalNAc and Gal as major constituents, and Man and GlcNAc as the minor ones. The presence of several N-linked chains was corroborated by studies with the inhibitor of N-glycosylation, tunicamycin and N-glycosidases. HPLC-analysis of glycans liberated after enzymatic (Oglycanase) or chemical (alkaline hydrolysis) cleavage showed that major type of O-glycans is core-1 mucin-type disaccharide Gal(ß01-3)GalNAc linked to Ser or Thr residues. Thus we consider the glycoprotein under study to be a first representative of mucin-type glycoprotein family in insects and propose to name it "mucinD". An in vitro examination of the purified mucinD performed by electron microscopy revealed that, similarly to vertebrate mucin glycoproteins, mucinD possesses a rod-like structure with no detectable globular domains present.
Monoclonal antibodies raised against mucinD were shown to recognize an epitope including Gal(ßl-3)GalNAc as an important part. Using these antibodies accumulation of mucinD was observed in the culture medium during cultivation of Drosophila cells. This process was blocked by secretion inhibitors, monensin and brefeldin A. The latter drug caused an accumulation in the cultured cells of incompletely glycosylated forms of mucinD, thus indicating that certain steps of the mucinD O-glycosylation pathway are localized to the trans-Golgi network compartments.
Subcellular immunolocalization of the epitope recognized by the monoclonal antibodies in mucinD revealed its presence in storage or secretion granules in a variety of tissues. Of specific interest was the finding of the mucinD epitope in ring canals - organelles involved in transport of macromolecules from trophocytes to a growing oocyte in developing egg chambers. The immunostaining can be observed even at the very early stages in region 1 of germarium when none of the known ring canal components (kelch-protein, profilin, actin) are recruited to this specific organelle. The antibodies can also reveal numerous ring canals connecting dividing primary spermatocytes in larval and adult testes. This seems to be the first case of immunolocalization of a ring canal component in animal testes. Further studies of this peculiar antigen is needed to elucidate its possible biological role during insect gametogenesis.
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