NCI, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NCI domain of the ctl(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NCl domain to be deduced as well as the C-terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.The main collagenous component of basement membranes is collagen IV. The triple helical molecule is 400 nm long and carries a globular domain at its carboxy-terminal [l -31. It contains two ctl (IV) chains and one a2(IV) chain, each consisting of approximately 1700 amino acid residues [4, 51. The triple-helical part of these chains is frequently interrupted by non-helical regions [6, 71, in contrast to interstitial collagen chains. In basement membranes, collagen IV forms a macromolecular network in which the 30-rim-long N-terminal region (7s domain) of four molecules are linked together. Each of these molecules becomes connected at the opposite end, via the C-terminal globule (NCI domain), to the NCI domain of yet another molecule. The network is stabilized by the formation of intermolecular cross-links at both ends [8, 91. This organization differs from the assembly of the interstitial collagens I, I1 and 111, whose 300-nm-long molecules are aligned in parallel but in a D-staggered array (D = 67 nm)While the amino acid sequence of large parts of the triplehelical region of the al(1V) chain from human [7] and mouse [6] is known, the C-terminal globular domain proved to be difficult to characterize, probably because of its hydrophobic character. Since the NCI domain is important for the assembly of the molecules, we decided to determine its amino acid sequence via the corresponding cDNA. Appropriate for such investigations is poly(A)+ RNA from tissues and cell lines which are known to produce basement membranes and type IV collagen, in particular the EHS mouse tumor [l I], the PYS-2 cell line [I, 121 and 1101.both sources contain very little translatable mRNA for the chains of type IV collagen, the amount being estimated to be less than 0.1 % (M. Laurent, I. Oberbaumer, unpublished). However, induced F9 cells have been reported to contain higher levels of these mRNAs [I 51.Here we will present our data on three cDNA clones, two from an EHS library and one from an F9 library, which cover the C-terminal area of the triple helix, the globular domain of the a1 (IV) chai...
The sequence of 511 residues from the C-terminal portion of the triple helix of mouse a2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an EngelbrethHolm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly-Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the a1 (IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the oll(IV) and a2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triplehelical sequences of the two constituent chains of basement membrane collagen.The collagenous constituent of basement membrane is collagen IV. Its molecule has the shape of a flexible thread with a length of 400 nm carrying a globular domain at the C terminus. Its macromolecular organization is a non-fibrillar network [l -31. The polypeptide chains of collagen IV, al(1V) and a2(IV), each contain about 1700 amino acid residues and are folded into three main segments, the triple-helical N-terminal cross-link (7s) domain (about 130 residues) [4], the central triple-helical segment (about 1300 residues) and the globular non-collagenous C-terminal (NC1) domain (229 residues) [5]. The two terminal domains are the important interaction sites for the formation of the macromolecular extracellular network of collagen IV. Four molecules interact via the N-terminal triple-helical cross-link domain overlapping one another by 30 nm, whereas only two molecules react via the NC1 domains. The central triple helix, which connects the interacting terminal sites, is thought to determine the biomechanical properties of the network.In contrast to the fiber-forming collagens which have a continuous triple helix, the central domain of the collagen IV molecule is frequently interrupted by non-triple-helical regions characterized by an imperfect Gly-Xaa-Yaa sequence repeat [6 -81. These interruptions appear to be important structural elements which modulate the flexibility of the molecule. This structural principle was first discovered in collagen IV, but is also found in other collagen types, such as collagen IX [9].Several studies have contributed to the complete elucidation of the primary structure of the al(1V) chain. The amino MATERIALS AND METHODS Protein chemicul investigationsFragment P2 ( M , about 55000) was purified from a pepsin digest of collagen IV obtained from mouse EHS tumor as previously described [12]. In addition, the digest contained a shorter variant P2* of M , about 20000, which was separated from P2 on CM-cellulose and purified on Sephadex G-75 to electrophoretic homogeneity (not shown). Fragment P2 was cleaved with cyanogen bromide and trypsin. The cyanogenbromide-derived peptide CB8 was further cleaved with SV8 protease. Conditions of cleavage and purification on ...
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