The basement membranes of nematodes and vertebrates are composed of similar protein components including laminin, perlecan, type IV collagen, and SPARC. The sequence conservation of these proteins suggests that basement membrane structure and function are conserved. To determine the extent of the structural similarities between C. elegans and vertebrate SPARC proteins, we have raised polyclonal antibodies against a bacterial fusion protein containing one of the calcium binding domains of nematode SPARC. Immunoblots of whole nematode lysates and of NP40 soluble proteins showed that nematode SPARC is a 27 kD protein (1). When electrophoresed under nonreducing conditions, SPARC shows increased mobility demonstrating that it has compact, disulfidebonded structure. Similarly, treatment with N-glycosidase F, which removes complex carbohydrates, resulted in increased mobility. Therefore, SPARC is glycosylated, probably on the conserved asparagine residue in the cysteine-rich domain. Together these biochemical analyses show that nematode SPARC is structurally very similar to its vertebrate counterparts. In mammalian cell culture, excess SPARC protein has been shown to reverse cell adhesion and cause cell rounding. Overexpression of wild type SPARC in nematodes causes body deformities and paralysis which could result from defects in muscle cell adhesion (2). To determine the effects of SPARC on muscle cell structure, a transgenic line was generated that carries extra copies of the SPARC gene (ost-l) along with the rol-6(d) gene N2;Ex[ost-1;rol-6(d)]). L4 larvae from this line develop an Unc phenotype with a protruding vulva, paralysis, and sterility. These animals were stained with fluorescently labeled phalloidin to visualize actin filament organization. While wild type animals form distinct sex muscle cell structures that attach at the vulva, the N2;Ex[ost-l;rol-6(d)] animals show no actin organization in their sex muscle cells. We conclude that the vulval protrusion observed in the presence of excess SPARC results from improper cytoskeletal organization of the sex muscle cells. As actin filament structure is dependent on cell adhesion, these results support the idea that in these transgenic animals SPARC is disrupting muscle cell adhesion. 1. Schwarzbauer, J.E., F. MussetBilal, and C.S. Ryan (1994) The extracellular matrix associated protein SPARC/osteonectin in Caenorhabditis elegans. Meth. Enzymol. 245, 257-270. 2. Schwarzbauer, J.E. and C.S. Spencer (1993) The Caenorhabditis elegans homolog of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol. Biol. Cell 4, 941- 952.

Affiliation:
- Dept. of Molecular Biology, Princeton University, Prineeton NJ 08544