Amino Acid Sequence and Domain Structure of Entactin. Homology with Epidermal Growth Factor Precursor and Low Density Lipoprotein Receptor

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'-untranslated region of 2.2 kb. The open reading frame encodes a 1,245-residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asnlinked glycosylation sites, and two potential Ca 2÷binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells. B ASEMENT membranes are a type of extracellular matrix that form thin sheets separating epithelial, endothelial, muscle, fat, and nerve cells from connective tissue (51). Epithelial cells require contact with a basement membrane to maintain their morphology and differentiated phenotype, and this is a result of interactions between basement membrane molecules and cell surface receptors for them (26). The major components of basement membranes are type IV collagen, laminin, entactin, and heparan sulfate proteoglycan (reviewed in reference 48). Entactin is a 150-kD sulfated glycoprotein first identified as a product of a teratocarcinoma-derived parietal endoderm line (6). It is identical to nidogen, a polypeptide originally isolated as an 80-kD proteolytic fragment from the Engelbreth-Holm-Swarm tumor (49). Immunostaining has shown entactin to be a ubiquitous component of adult, fetal, and extraembryonic basement membranes (4, 6, 17, 29, 37, 48, 54), and it is also synthesized by early embryos (17, 54), embryonal carcinoma cells (7, 11, 17), and mesenchyme cells (28, 29, 52). Laminin and entactin can be coextracted from cell culture media and basement membranes in the form of a highly stable noncovalent complex (7, 17, 32, 40). As visualized by electron microscopy, the complex consists of one entactin molecule bound to one of the short arms of laminin, near the center of the cross (40). Entactin also binds to fibronectin and type IV collagen, but has no affinity for heparan sulfate proteoglycan (18). Progress in elucidating the function ofentactin has been slow, due to its susceptibility to proteolysis during extraction and the fact that denaturants required to inhibit proteases and dissociate the laminin-entactin complex cause a loss of binding activity (18, 40). We have used cDNA cloning to obtain the complete amino acid sequence of mouse entactin, and this has provided new insights into the structure, function, and evolution of the molecule. The entactin sequence has been found to contain EGF-like cysteine-rich repeats, segments showing homology to the EGF precursor, the low density lipoprotein (LDL) ~ receptor, and thyroglobulin, and possible sites for Ca 2+and cell binding. Materials and Methods Purification and NHz-terrainal Amino Acid Sequencing of Entactin The extracellular matrix proteins synthesized by the mouse parietal endoderm cell line M1536-B3 were extracted as previously described (10), and resolved by SDS-PAGE (35) on 5% polyacrylamide slab gels. The bands were visualized by soaking the gels in 1 M KCI (24). The entactin bands were cut out and the protein isolated by electroelution (30). The preparation was homogeneous when tested by SDS-PAGE. Samples were dialyzed against three 2.000-vol changes of H20 over a 36-h period, and NH2terminal sequence analysis of purified entactin was performed on an amino acid sequencer (model 890M; Beckman Instruments, Inc., Palo Alto, CA). 1. Abbreviation used in this paper: LDL, low density lipoprotein. © The Rockefeller University Press, 0021-9525/88/12/2749/8 $2.00 The Journal of Cell Biology, Volume 107, (No. 6, Pt. 2) Dec. 1988 2749-2756 2749 on Jne 7, 2017 D ow nladed fom Published December 1, 1988