A Molecular Analysis of Hyalin—A Substrate for Cell Adhesion in the Hyaline Layer of the Sea Urchin Embryo

Abstract

The hyaline layer of echinoderm embryos is an extraembryonic matrix that functions as a substrate for cell adhesion through early development. The major constituent of the hyaline layer is the protein hyalin, a fibrillar glycoprotein of approximately 330 kDa that multimerizes in the presence of calcium. Here we provide a molecular characterization of hyalin and identify a region of the protein that is important for its function in cell adhesion. Partial hyalin cDNAs were identified from two sea urchin species, Strongylocentrotus purpuratus and Lytechinus variegatus, by screening expression libraries with monoclonal antibodies to hyalin. The cDNAs each encode a tandemly arranged series of conserved repeats averaging 84 amino acids. These hyalin repeats are as similar between the two species as they are to repeats within each species, suggesting a strong functional conservation. Analysis of this repeat shows that it is a unique sequence within the GenBank database with only weak similarity to mucoid protein sequences. The hyalin mRNA is approximately 12 kb in length and is present in developing oocytes coincident with the appearance of cortical granules, the vesicle in which the hyalin protein is specifically packaged. The mRNA is present throughout oogenesis but is rapidly lost at oocyte maturation so that eggs and early embryos have no detectable hyalin mRNA. The hyalin protein, however, remains at relatively constant levels throughout development. Thus, all the hyalin protein present during early development, when no RNA is detectable, is maternally derived and exocytosed from cortical granules at fertilization. Hyalin mRNA reaccumulates in embryos beginning at the mesenchyme blastula stage; a RNA gel blot and in situ hybridization analysis of gastrulae and larvae shows a progressive confinement of hyalin mRNA to the aboral ectoderm. Recombinant hyalin containing the tandem repeat region of the protein was expressed in bacteria and is shown to serve as an adhesive substrate, almost equal to that of native hyalin, in cell adhesion assays. This adhesive activity is partially blocked by dilute hyalin monoclonal antibody Tg-HYL to the same extent as that for native hyalin. Thus, this hyalin repeat region appears to contain the ligand for the hyalin cell surface receptor. These data help explain some of the classic functions ascribed to the hyalin protein in early development and now enable investigators to focus on the mechanisms of cell interactions with the hyaline layer.

Exogenous hyalin and sea urchin gastrulation, Part II: hyalin, an interspecies cell adhesion molecule

“…The hyalin repeat domain is thought to be a distinct superfamily within the immunoglobulin-like fold and is believed to function in cell adhesion (Wessel et al, 1998;Callebaut et al, 2000).…”

Hyalin is a cell adhesion molecule involved in mediating archenteron–blastocoel roof attachment

“…In a single cell assay, using cells dissociated from L. variegatus sea urchin embryos, it was found that cells bound to both native hyalin or a bacterially expressed recombinant hyalin repeat, which does not contain carbohydrate (Wessel et al, 1998). An anti-hyalin monoclonal antibody (Tg-HYL (McAb183) blocked cell binding to both native and recombinant hyalin (Wessel et al, 1998).…”

“…An anti-hyalin monoclonal antibody (Tg-HYL (McAb183) blocked cell binding to both native and recombinant hyalin (Wessel et al, 1998). This same antibody caused cells in vivo to retract from the hyaline layer, blocking gastrulation (Adelson and Humphreys, 1988).…”

“…It serves as an adhesive substrate during early development (Herbst, 1900;McClay and Fink, 1982;Wessel et al, 1998) and consists of repeated regions (called hyalin repeats) averaging 84 amino acids (Wessel et al, 1998;Callebaut et al, 2000) and non-repeated regions (Wessel et al, 1998). The Gen Bank database suggests that the hyalin repeat is a unique sequence that shows slight similarity to mucoid protein sequences (Callebaut et al, 2000) and appears to be related to the immunoglobulin-like fold (Callebaut et al, 2000).…”

“…The Gen Bank database suggests that the hyalin repeat is a unique sequence that shows slight similarity to mucoid protein sequences (Callebaut et al, 2000) and appears to be related to the immunoglobulin-like fold (Callebaut et al, 2000). Since hyalin consists of only 2-3% carbohydrate (Citkowitz, 1971), it is not very similar to mucins that contain more carbohydrate (Wessel et al, 1998). The hyalin repeat has been identified in bacteria, mice, Caenorhabditis elegans, and Drosophila melanogaster proteins, as well as in a human protein (Callebaut et al, 2000).…”