Worm Breeder's Gazette 5(2): 16
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.
Studies on vertebrate collagens have revealed that pepsin, at low temperature and pH- specifically digests non-helical regions (usually at NH2 and COOH termini) of native collagen, but does not digest triple helical regions. Since C. elegans cuticles contain 70% collagen (by weight), we decided to investigate the effects of limited pepsin digestion. Our procedure for isolating cuticle proteins involves 3 steps: first, sonication of whole animals in a low osmotic strength buffer; second, boiling these crude cuticles in 1% SDS for 2 min to remove all non- cuticle material; and third, solubilization of cuticle proteins with - mercaptoethanol in the presence or absence of detergent. In separate experiments cuticle proteins were exposed to pepsin at each of these three steps: After sonication but before incubation with SDS; after incubation with SDS followed by extensive washing to remove detergent; after extraction with -mercaptoethanol in a neutral salt solution. With either sonicated or SDS-cleaned cuticles, cuticle proteins were not solubilized by pepsin but were solubilized after subsequent reduction of S-S bonds with -mercaptoethanol. SDS-PAGE of the solubilized proteins separated them into 8 bands (MW range: 40K-125K). These bands were identical in sonicated and in SDS-cleaned cuticles suggesting that SDS does not irreversibly denature the proteins. In controls without pepsin the usual cuticle protein pattern was observed: 8 bands with molecular weights between 54K and 212K and large numbers of higher molecular weight bands which account for 20% of the soluble proteins. No high molecular weight material was present in gels of pepsin-treated proteins suggesting that the high molecular weight is composed of the low molecular weight (54K-212K) species crosslinked within the non-helical regions. Pepsin digestion of these regions presumably converts them into the eight 40K-125K pepsin resistant proteins. In addition, if pepsin actually degrades all nonhelical material, these experiments indicate that S-S bonds within helical regions are responsible, at least in part, for crosslinking of collagens within the cuticle since -mercaptoethanol is still required to solubilize the pepsin resistant material. In a third type of experiment, SDS was washed out of cleaned cuticles and proteins were extracted with -mercaptoethanol into a neutral salt solution and then exposed to pepsin in order to see if we could extract native helices that are pepsin resistant when not attached to the cuticle. Resistance to pepsin degradation was used as a criterion for helix stability. SDS-PAGE of these digestion products gave 7 bands (MW 39K-110K). The molecular weights and relative amounts of these proteins appeared similar but not identical to those obtained when in situ cuticle proteins were exposed to pepsin. In all three types of experiments 2 very prominent major bands were obtained ( MW 67K and 81K). Non-pepsin-treated control cuticle extracts also contain 2 very prominent bands (MW 91K and 106K). It is tempting to speculate that pepsin simply degrades the non-helical termini of these proteins to create the two lower molecular weight bands. If this is true, then approximately 25% of each of these collagen chains is not in a triple helical conformation. This figure is in good agreement with estimates of the maximal possible amount of triple helix (78%) from determinations of the glycine content of cuticle collagens (Cox, Kusch and Edgar, manuscript submitted). The pepsin resistance of solubilized cuticle proteins demonstrates that native collagen helices can be extracted from cuticles by reduction of S-S bonds. Although interchain S-S bonding occurs within helical regions, presumably this crosslinking is not necessary for maintenance of native helices. These experiments indicate that C. elegans cuticle collagen like vertebrate collagens, is in a triple helical conformation. In C. elegans, however, these helices are cross-linked by S-S bonds. Molecular weights of the pepsin-resistant polypeptides suggest that a single polypeptide is not folded upon itself to form a triple helix, as has been suggested for Ascaris cuticle collagen