A six-armed complex could be extracted from the marine sponge Osccrrellu tuberculuta by a two-step incubation, first in Tris-buffered saline containing EDTA, then in Tris-buffered saline containing urea. The crude extracts contained, in addition, collagen fibrils with surface filaments, individual filaments resembling collagen molecules, and laminidnidogen-like complexes. The extracts were subsequently purified by gel-filtration chromatography and low-pressure ion-exchange chromatography on DEAE-cellulose, then analyzed by SDS/PAGE and iinmunoblotting methods. A glycoprotein of high molecular mass was isolated, and reduced to subunits of 230 kDa. After transfer to nitrocellulose, both the complex and its subunits were faintly stained by antibodies against amphibian tenascin. Electron microscopy of the purified extracts demonstrated the presence of a large population of tenascin-like molecules and complexes of several molecules interacting with each other by their central globule.Tenascin (also known as hexabrachion, cytotactin, J1, myotendinous antigen) is a large extracellular-matrix glycoprotein containing six similar disulfide-linked subunits which give the protein a characteristic six-armed structure when visualized by electron microscopy ([l-31 for review). It has a distinctive tissue distribution during development [4, 51 and several reports described both its adhesive and anti-adhesive properties (reviewed in [6, 71). Recent studies indicate that the promoter of the tenascin gene contains target regions responding to homeodomain proteins, suggesting that tenascin could be a morphoregulatory molecule 18, 91. However, the crucial role of tenascin during development has been questioned by recent data. Homologous recombination experiments produced mutant mice in which tenascin gene expression was disrupted, but the homozygous null-mutant mice had apparently no defect [lo], at least under resting conditions.Tenascin has been characterized in several vertebrate groups, including Amphibians [ll]. Its presence has been also demonstrated in leech, where it promotes neurite outgrowth [12, 131. Tenascin-like proteins have been localized in sea-urchin embryos [14], and two genes related to tenascin have been detected in Drusophilu: Ten' [15] and Ten"' (Baumgartner, S . , unpublished results).In the course of our research program on the evolution of the extracellular matrix, we have investigated the presence of basement-membrane molecules in Porifera. The Porifera constitute the most primitive phylum of multicellular animals. They possess an extended extracellular matrix [16] containing at least two types of collagen [17-191, However, a group of marine sponges, the Homosclerophorida, possess laminae underlying their epithelia which resemble basement membranes [23]. These sponges have a simplified organization which is sometimes considered as a result of an evolutionary process [24]. Pieces of Oscurellu where treated in order to extract laminin, an adhesive glycoprotein of basement membranes, according to an efficie...
Summary— Snail muscles were extracted by a solution of EDTA and electron microscopy showed that the extract contained dispersed, depolymerized collagen fibrils and cross‐shaped laminin‐like structures. The extracts were purified by ultracentrifugation followed by two different procedures which enriched the content of laminin‐like structures. The laminin‐related molecules displayed unique properties when analyzed by biochemical, immunological and morphological methods. Electrophoretic patterns of the molecular form purified primarily by ion exchange chromatography, resembled EHS‐tumor laminin and displayed a cruciform shape when viewed by electron microscopy. Immunohistology, using antiserum obtained against the agarose gel‐purified protein, showed that this laminin was primarily located in the extracellular matrix surrounding muscle fibers. Western blots using anti‐EHS laminin antibody showed reaction of a 300 kDa subunit of this snail laminin. The protein obtained by another procedure, initially using gel filtration, followed by ion exchange chromatography, also appeared to be a laminin. It had a collapsed cruciform appearance when viewed by electron microscopy. It contained several different subunits, one of which, ca 300 kDa, reacted with anti‐EHS‐laminin antibody and with anti‐snail laminin antibody. In contrast, EHS laminin did not react with the anti‐snail laminin antibody. The composite results suggest that at least two different forms of laminin are extractable from snail muscle and that they share molecular properties and immune determinants with mouse tumor laminin.
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