Worm Breeder's Gazette 8(3): 57
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Affinity purified cathepsin D from C. elegans shows 5 (sometimes 6) distinct bands on SDS gels, ranging from 32 kDa to 40 kDa. We designate the bands as 1 (40 kDa) through 5 (32 kDa). Bands 3 and 4 constitute the great majority of the enzyme both in crude extracts and in purified preparations. We now have reason to believe that these bands represent variations on the same parent protein, differing from each other principally in the nature of the attached oligosaccharide(s) . We have made peptide fingerprints of bands 1, 3, 4 and 5 by reduction of the disulfide bonds, carboxamidation, iodination with [125I], resolution of the bands on SDS gels and tryptic fingerprinting of the excised gel bands. [Without reduction, cathepsin D is utterly insensitive to trypsin.] There is evidently a high degree of homology among the multiple forms, but resolution is not yet high enough to be sure that there are not some differences in the polypeptide chains, even for the limited subset of tyrosine-containing peptides. Periodate-Schiff staining of SDS gels shows that all five bands contain carbohydrate, but probing SDS gel electroblots with labeled lectins reveals differences between the oligosaccharides. Iodinated lentil lectin (mannose specificity) binds to band 1 and more weakly to bands 3 and 4, whereas ConA (also mannose specificity) binds principally to band 2. Wheat germ agglutinin (chitobiose specificity) binds principally to band 1. In all cases, binding is competitively inhibited by the appropriate free glycosides. Band 3 specifically binds anti-HRP Ab (kindly provided by Shahid Siddiqui and Joe Culotti), which also binds to ovalbumin. We suspect that this polyclonal anti- HRP recognizes oligosaccharide determinants. Slab gel isoelectric focusing, followed by immunoblotting with anti-cathepsin D Ab, shows that there are two distinct isoelectric forms of cathepsin D. Bands 3, 4 and 5 have pI=5.2 and band 1 appears to have pI=4.9. (Band 2 was weak or absent in this particular preparation.) Our inference is that bands 3, 4 and 5 are likely to contain the same polypeptide chain, differing from each other in content of neutral sugar. The additional anionic groups on band 1 may represent additional polypeptide, sialic acid, mannose-phosphate (as in mammalian lysosomal enzymes) or sulfated oligosaccharide (as in Dictyostelium lysosomal enzymes). The purified cathepsin D from a cad-1(j001) mutant has the same molecular weight distribution and isoelectric points as the wild-type enzyme. Thus, the j001 mutation does not alter charged amino acid residues.