Worm Breeder's Gazette 12(3): 53 (June 15, 1992)

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.

Structure and Expression of the C. elegans Homolog of the Mammalian "Anti-Spreading" Protein, SPARC

Jean E. Schwarzbauer, Carol S. Spencer

Department of Molecular Biology, Princeton University, Princeton, NJ 08544

The interaction of cells with the surrounding tissue extracellular matrix is essential for normal cell behavior and morphology in all organisms. In response to specific stimuli, cells strengthen or weaken their matrix associations thus modulating cell shape. One protein that affects cell morphology in culture systems is the "anti-spreading" protein, SPARC (Secreted Protein Acidic and Rich in Cysteine) ( 1). SPARC is a secreted, C a2 +bindingprotein that is expressed at high levels in bone, parietal endoderm, and other tissues with high rates of cell proliferation and remodeling. In vitro, SPARC induces rounding of attached and spread cells (2).

We have identified the C. elegans homolog of mammalian SPARC. cDNA and genomic DNA clones have been isolated. The 3.6 kb gene has five introns that correspond in position to five of the nine introns in the mouse SPARC gene. The cDNA is 1.1 kb containing a 795 bp open reading frame that shows 55% identity to the mouse SPARC nucleotide sequence. The SPARC gene is located near the end of chromosome IV(L), to the right of daf-1 (A. Coulson, personal communication).

SPARC can be divided into four domains based on predicted protein structure (3). The acidic domain I and a-helical domain m are significantly shorter in nematodes than mice and show only very limited homology. Domain II has a conserved cysteine motif that is homologous to several protease inhibitors (3) and to a repeating structure in the neural basement membrane protein, agrin (4). Finally, the highly conserved domain IV is a C a2 +binding,EF-hand structure that also has anti-spreading activity, and binds to several extracellular matrix proteins. Based on the homology, we predict that nematode SPARC will also be localized to extracellular matrix and will bind C a2 +.

To identify the tissues responsible for expression of SPARC in the nematode, we prepared transgenic animals with a SPARC-lacZ fusion construct. A cassette from the plasmid, pPD22 .04,containing the lacZ gene plus a nuclear localization signal (5), was inserted in frame into the first exon thus maintaining all sequences upstream and within the SPARC gene. The resulting construct was injected along with the rol-6 dominant marker, and lines established from individual transgenic animals were stained with X-gal. SPARClacZ staining was confined to body-wall and vulval muscle cells and developed very rapidly, within 5-10 min, indicating high level expression from the SPARC promoter.

The expression of SPARC by body-wall muscle suggests that this secreted protein affects the adhesion and morphology of nerve, muscle, or hypodermal cells by associations with the adjacent basement membranes. Protein localization studies are beginning as is a screen for mutants within the SPARC gene.

We are indebted to Iva Greenwald for her invaluable advice, interest, and support throughout the course of this work, and we also thank Alison Januzzi for preparing the SPARC-lacZ transgenic worms.

Literature Cited:

(1) Termine et al. (1981) Cell 26, 99-105; Mason et al. (1986) EMBO J. 5, 1465-1472.

(2) Sage et al. (1989) J. Cell Biol. 109, 341-356.

(3) Engel et al. (1987) Biochemistry 26, 6958-6965.

(4) Rupp et al. (1991) Neuron 6, 811-823.

(5) Fire et al. (1990) Gene 93, 189-198.