Identification of a component of the sea urchin hyaline layer, HLC-175, which undergoes proteolytic processing during development.
To define the role(s) played by the sea urchin extraembryonic matrix, the hyaline layer, we have previously purified and characterized a number of the protein components of this structure. We are currently studying the timing and significance of the proteolytic processing of these species. The localization of HLC-175 in the egg and 1-hr-old embryo was determined by indirect immunofluorescence analysis. The relationship between HLC-175 and the 109- and 81 kDa species was determined by a combination of native gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and protein gel blot analyses using the anti-175, -109 and 81 kDa antisera. Using gel exclusion chromatography we have fractionated a mixture of proteins extracted from the surface of 1-hr-old sea urchin embryos. A set of fractions eluting from the column contained three species of apparent molecular masses 175-, 109- and 81 K. These species comigrated on analysis by either non-reducing SDS-PAGE or native gel electrophoresis. Inclusion of the reducing agent, dithiothreitol, in the solubilizing solutions abolished comigration of these polypeptides. When polyclonal antisera were prepared against each of these antigens cross-reactivity between the 175- and 109 kDa species and between the 175- and 81 kDa species was detected. Developmental protein gel blot analyses revealed a precursor-product relationship between the 175- and the 109- and 81 kDa polypeptides. Indirect immunofluorescence analysis confirmed the localization of HLC-175 to the hyaline layer. The results reported here clearly identify HLC-175 as a component of the hyaline layer.(ABSTRACT TRUNCATED AT 250 WORDS)
Robinson
J J
JJ
Department of Biochemistry, Memorial University of Newfoundland, St John's, Canada.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003429:Cross Reactions; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008970:Molecular Weight; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011499:Protein Processing, Post-Translational; D012617:Sea Urchins / Q000737:chemistry* / Q000378:metabolism
D000906:Antibodies; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; C076585:hyalin protein, Echinoidea; D004229:Dithiothreitol