Cuticular localisation and turnover of the major surface glycoprotein (gp29) of adult Brugia malayi.
A polyclonal antiserum was raised to a gel purified preparation of the major water-soluble surface glycoprotein (gp29) of adult Brugia malayi, and used to define the stage specificity of expression, localisation (by immunoelectron microscopy) and the dynamics of biosynthesis and turnover via pulse-chase experiments. Gp29 was not detected in surface-labelled preparations of either pre- or post-parasitic third stage larvae (L3), but was present in fourth stage larvae (L4), where its mass was estimated to be 30 kDa by SDS-PAGE. In both L4 and adult worms, the protein resolved as 3 distinct species in 2-dimensional electrophoresis, with pIs from 6.5 to 7.5. Pulse-chase studies via metabolic labelling of adult worms with [35S]methionine in vitro indicated that gp29 was processed from a 32-kDa precursor to the mature molecule within 45 min and that it was secreted into culture medium within 5 h of synthesis. On extended culture, gp29 was converted to a 56-kDa product, presumably either by complex formation or covalent linkage with another secreted molecule. This higher molecular weight component had a more acidic pI of 4.5 and was insensitive to digestion with N-glycanase. Immunoelectron microscopy showed that gp29 was distributed throughout the cuticle and hypodermal cell layer of adult worms, suggesting that the protein was synthesised in the hypodermis, and that turnover into culture medium occurred through the cuticle. The protein appeared to concentrate at the distal cell membrane of the hypodermis, particularly at the stacked invaginations. Additional immunostaining was found on the basement membrane of the basal lamina of the intestine.
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, U.K.
M E
ME
Selkirk
W F
WF
Gregory
M
M
Yazdanbakhsh
R E
RE
Jenkins
R M
RM
Maizels
D000818:Animals; D002009:Brugia / Q000648:ultrastructure*; D005260:Female; D007814:Larva / Q000648:ultrastructure; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis*; D016253:Microscopy, Immunoelectron; D051381:Rats; D013194:Staining and Labeling
D008562:Membrane Glycoproteins