Immunodetection of phosphotyrosine-containing proteins in the integument, fat body and haemocytes of the mediterranean fruit-fly Ceratitis capitata: Possible involvement in pupariation

Abstract

Antibodies raised against the synthetic hapten $\text{p-}\text{azobenzyl}$ phosphonate (ABP), which specifically cross react with phosphotyrosine, were used to detect phosphotyrosine-containing proteins in integument, fat body, and haemocytes of C. capitata white pupae. Immunofluorescence experiments demonstrated that fat body, integument, and haemocytes contained proteins phosphorylated in tyrosine residues, while other tissues, such as muscle, showed no fluorescence. To determine which polypeptides are recognized by ABP antibodies, an immunoblotting analysis was performed. The results revealed that the majority of the immunoreactive polypeptide with a $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ ranging between 35- and 130 kDa were detected in the integument of C. capitata. Three immunoreactive polypeptides seem to be common in tissues we have examined. 130 kDa is common in integument and fat body, 98 kDa in integument and haemocytes, and 93 kDa is common in haemocytes and fat body. Solubilization of integumental proteins in Tris-HCl buffer pH 7.2 containing either KCl, or NaCl, or urea, or swittergen shows that several phosphotyrosine-containing proteins form non-covalent links with other cuticular components and/or cytoskeleton. Furthermore, treatment of unsolubilized fraction with lysozyme, indicated that some N-acetylglucosamine-containing material prevents extraction with salt solution; this material may be chitin or some intracellular material. The possible contribution of phosphotyrosine containing proteins in the pupariation process is discussed.