Characterization of a 24 kD parasitism-specific hemolymph protein from pharate pupae of the caribbean fruit fly,Anastrepha suspensa

A parasitism-specific protein was originally identified in the hemolymph of the Caribbean fruit fly Anastrepha suspensa parasitized by the braconid wasp Diachasmimorpha (Biosteres) longicaudata using single-dimensional (1-D) sodium dodecyl sulfate (SDS) PAGE. We now show that the protein is comprised of two closely migrating species both of which are glycoproteins of ≈? 24,000 Daltons (24 kD). The proteins were poorly resolved from whole hemolymph by 1-D SDS PAGE, but were well resolved by two-dimensional (2-D) PAGE and isoelectric focusing. They have pl's of ≈? 6.3 and 6.7 and contain Man residues, based on their affinity for concanavalin A (Con A). The presence of GlcNAc, NeuAc, and GalNAc residues in both proteins was implicated by their binding to wheat germ agglutinin (WGA). The proteins bound WGA more intensely following mannosidase treatment which eliminated their affinity to Con A and further implicated the presence of internal GlcNAc residues. However, binding of the proteins to WGA in the presence of competing GlcNAc (1 M) was reduced but not eliminated and suggested that in addition to GlcNAc, other WGA-binding sugar moieties, possibly NeuAc, a Sia, were present. To evaluate the presence of NeuAc, we treated the hemolymph with Vibrio cholerae neuraminidase which specifically cleaves terminal Sia. Samples of the neuraminidase-digested proteins were evaluated by WGA binding and Western blotting with the use of an anti-24 kD rabbit polyclonal serum to determine whether desialation eliminated the proteins' affinity to WGA or their immunoreactivity. Our results show that partial digestion of the 24 kD proteins with Vibrio cholerae neuraminidase resulted in two immunoreactive bands in Western blots of 1-D gels but only one of these, the upper undigested 24 kD band, bound WGA. This confirmed the presence of Sia residues in the proteins and demonstrated that desialation increased their relative electrophoretic mobilities. © 1994 Wiley-Liss, Inc.