Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.     

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.     

Substitutions for glycine alpha 1-637 and glycine alpha 2-694 of type I procollagen in lethal osteogenesis imperfecta. The conformational strain on the triple helix introduced by a glycine substitution can be transmitted along the helix

Two substitutions for glycine in the triple-helical domain were found in type I procollagen synthesized by skin fibroblasts from two probands with lethal osteogenesis imperfecta. One was a substitution of valine for glycine alpha 1-637, and the other was a substitution of arginine for glycine alpha 2-694. The effects of the mutations on the zipper-like folding of the collagen triple helix were similar, since there was post-translational overmodification of the collagenase A fragments (amino acids 1-775) but not of more COOH-terminal fragments of the protein. The mutations differed markedly, however, on their effects on thermal unfolding of the triple helix. The collagenase A fragment from the collagen containing the arginine alpha 2-694 substitution was cleaved at about amino acid 700 when incubated with trypsin at 30-35 degrees C. Therefore, there was micro-unfolding of the triple helix at a site close to the glycine substitution. Surprisingly, however, the collagenase A fragment with the valine alpha 1-637 substitution was also cleaved at about amino acid 700 under the same conditions. The results, therefore, demonstrated that although most glycine substitutions delay folding of the triple helix in regions that are NH2-terminal to the site of the substitution, the effects on unfolding can be transmitted to regions that are COOH-terminal to the site of the glycine substitution.     

Substitution of serine for alpha 1(I)-glycine 844 in a severe variant of osteogenesis imperfecta minimally destabilizes the triple helix of type I procollagen. The effects of glycine substitutions on thermal stability are either position of amino acid specific

Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.     

A disease-associated glycine substitution in BP180 (type XVII collagen) leads to a local destabilization of the major collagen triple helix.

BP180 is a homotrimeric transmembrane protein with a carboxy-terminal ectodomain that forms an interrupted collagen triple helix. Null type mutations in the BP180 gene produce a recessive subepidermal blistering disease, non-Herlitz junctional epidermolysis bullosa. Like the null mutations, a glycine substitution (G627V) within the longest BP180 collagenous domain (COL15) is also associated with the recessive skin disease; however, unlike the null mutations, this glycine substitution appears to act in a dominant fashion to give rise to a novel form of random pitting dental enamel hypoplasia. The dominant effects of this mutation were thought to be due to alterations in the assembly and/or stability of this BP180 collagenous region. To further investigate this issue, a structural analysis was performed on recombinant forms of the wild type and G627V mutant BP180 ectodomain. Both proteins were found to form collagen-like triple helices with very similar Stokes radii and melting temperatures and exhibited very similar rates of synthesis, secretion and turn-over. Tryptic digestion analysis revealed that the mutant G627V-sec180e contains an additional highly sensitive proteolytic site that maps within the region of the mutation. Thus, the disease-associated G627V mutation in BP180 does not grossly alter protein structure, but causes a local destabilization of the triple-helix that exposes sensitive residues to the in vitro effects of trypsin and possibly affects its structure-function in vivo.     

Properties of the collagen type XVII ectodomain. Evidence for n- to c-terminal triple helix folding

Collagen XVII is a transmembrane component of hemidesmosomal cells with important functions in epithelial-basement membrane interactions. Here we report on properties of the extracellular ectodomain of collagen XVII, which harbors multiple collagenous stretches. We have recombinantly produced subdomains of the collagen XVII ectodomain in a mammalian expression system. rColXVII-A spans the entire ectodomain from deltaNC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains deltaNC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1. Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with ratios for hydroxyproline/proline of 0.4 and for hydroxylysine/lysine of 0.5. A small proportion of the hydroxylysyl residues in rColXVII-C ( approximately 3.3%) was glycosylated. Limited pepsin and trypsin degradation assays and analyses of circular dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the C-terminal rColXVII-D did not adopt a triple-helical fold. These results were further substantiated by electron microscope analyses, which revealed extended molecules for rColXVII-A and -C, whereas rColXVII-D appeared globular. Thermal denaturation experiments revealed melting temperatures of 41 degrees C (rColXVII-A), 39 degrees C (rColXVII-B), and 35 degrees C (rColXVII-C). In summary, our data suggest that triple helix formation in the ectodomain of ColXVII occurs with an N- to C-terminal directionality.     

Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Differential unfolding of alpha1 and alpha2 chains in type I collagen and collagenolysis

Collagenolysis plays a central role in many disease processes and a detailed understanding of the mechanism of collagen degradation is of immense interest. While a considerable body of information about collagenolysis exists, the details of the underlying molecular mechanism are unclear. Therefore, to further our understanding of the precise mechanism of collagen degradation, we used molecular dynamics simulations to explore the structure of human type I collagen in the vicinity of the collagenase cleavage site. Since post-translational proline hydroxylation is an important step in the synthesis of collagen chains, we used the DNA sequence for the α1 and α2 chains of human type I collagen, and the known amino acid sequences for bovine and chicken type I collagen, to infer which prolines are hydroxylated in the vicinity of the collagenase cleavage site. Simulations of type I collagen in this region suggest that partial unfolding of the α2 chain is energetically preferred relative to unfolding of α1 chains. Localized unfolding of the α2 chain leads to the formation of a structure that has disrupted hydrogen bonds N-terminal to the collagenase cleavage site. Our data suggest that this disruption in hydrogen bonding pattern leads to increased chain flexibility, thereby enabling the α2 chain to sample different partially unfolded states. Surprisingly, our data also imply that α2 chain unfolding is mediated by the non-hydroxylation of a proline residue that is N-terminal to the cleavage site in α1 chains. These results suggest that hydroxylation on one chain (α1) can affect the structure of another chain (α2), and point to a critical role for the non-hydroxylation of proline residues near the collagenase cleavage site.     

Identification of two distinct antibacterial domains within the sequence of bovine alpha(s2)-casein.

Two distinct domains with antibacterial activity were isolated from a peptic hydrolysate of bovine alpha(s2)-casein. The digested alpha(s2)-casein was fractionated by cation-exchange chromatography, after which the peptides in the two active fractions obtained were separated by high-performance liquid chromatography and sequenced by electrospray-ionization tandem mass spectrometry. The major component in each active fraction, f(183-207) and f(164-179), was further purified and the antibacterial activity of these components was tested against several microorganisms. Depending on the target bacterial strain, these peptides exhibited minimum inhibitory concentrations between 8 and 99 microM. Peptide f(183-207) exhibited a consistently higher antibacterial activity than f(164-179), although both peptides showed a comparable hemolytic effect. A method of in situ enzymatic hydrolysis on a cation-exchange membrane to obtain a fraction enriched in the most active antibacterial domain is presented. The antibacterial and hemolytic activities are discussed in relation to the structure and hydrophobicity of the peptides.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

The effect of the L-azetidine-2-carboxylic acid residue on protein conformation. IV. Local substitutions in the collagen triple helix

The properties of collagen are affected by the replacement of Pro by imino acid analogues. The structural effect of the low-level local substitution of L -azetidine-2-carboxylic acid (Aze) has been analyzed by computing the energy of CH₃CO-(Gly-Pro-Pro)₄-NHCH₃ triple helices in which a single residue of one strand has been replaced by Aze. When Aze is in position Y of a (Gly-X-Y) unit, low-energy local deformations are introduced in the triple helix, i.e., it becomes more flexible. On the other hand, the flexibility of the triple helix is not increased with Aze in position X. The energy of the triple helix to coil transition is not changed significantly by this amount of substitution. In an earlier study, we have demonstrated that the regular substitution of Aze in every tripeptide distorts or destabilizes the triple helix to a large extent [A. Zagari, G. Némethy, & H. A. Scheraga (1990) Biopolymers, Vol. 30, pp. 967–974 ]. Thus, it appears that a high level of substitution is required to cause the observed chemical and biological effects of Aze on collagen. © 1994 John Wiley & Sons, Inc.     

Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI

Collagen XI is a heterotrimeric molecule found predominantly in heterotypic cartilage fibrils, where it is involved in the regulation of fibrillogenesis. This function is thought to involve the complex N-terminal domain. The goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism. The amino-propeptide (alpha1-Npp) alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography. Cys-1-Cys-4 and Cys-2-Cys-3 disulfide bonds were detected by liquid chromatography-tandem mass spectrometry. This pattern is identical to the homologous alpha2-Npp, indicating that the recombinant proteins were folded correctly. Anomalous elution on molecular sieve chromatography suggested that the variable region was extended, which was confirmed using rotary shadowing; the alpha1-Npp formed a globular "head" and the variable region an extended "tail." Circular dichroism spectra analysis determined that the alpha1-Npp comprised 33% beta-sheet, whereas the variable region largely comprised non-periodic structure. Taken together, these results imply that the alpha1-Npp cannot be accommodated within the core of the fibril and that the variable region and/or minor helix facilitates its exclusion to the fibril surface. This provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis.     

Molecular mechanism of alpha 1(I)-osteogenesis imperfecta/Ehlers-Danlos syndrome: unfolding of an N-anchor domain at the N-terminal end of the type I collagen triple helix

We demonstrate that 85 N-terminal amino acids of the alpha1(I) chain participate in a highly stable folding domain, acting as the stabilizing anchor for the amino end of the type I collagen triple helix. This anchor region is bordered by a microunfolding region, 15 amino acids in each chain, which include no proline or hydroxyproline residues and contain a chymotrypsin cleavage site. Glycine substitutions and amino acid deletions within the N-anchor domain induce its reversible unfolding above 34 degrees C. The overall triple helix denaturation temperature is reduced by 5-6 degrees C, similar to complete N-anchor removal. N-propeptide partially restores the stability of mutant procollagen but not sufficiently to prevent N-anchor unfolding and a conformational change at the N-propeptide cleavage site. The ensuing failure of N-proteinase to cleave at the misfolded site leads to incorporation of pN-collagen into fibrils. Similar, but weaker, effects are caused by G88E substitution in the adjacent triplet, which appears to alter N-anchor structure as well. As in Ehlers-Danlos syndrome (EDS) VIIA/B, fibrils containing pN-collagen are thinner and weaker causing EDS-like laxity of large and small joints and paraspinal ligaments. However, distinct structural consequences of N-anchor destabilization result in a distinct alpha1(I)-osteogenesis imperfecta (OI)/EDS phenotype.     

A point mutation in a type I procollagen gene converts glycine 748 of the alpha 1 chain to cysteine and destabilizes the triple helix in a lethal variant of osteogenesis imperfecta

Synthesis of type I procollagen was examined in fibroblasts from a proband with a lethal perinatal variant of osteogenesis imperfecta. After trypsin digestion of the type I procollagen, a portion of the alpha 1 (I) chains was recovered as disulfide-linked dimers. Digestion of the protein with vertebrate collagenase and mapping of cyanogen bromide peptides suggested that a new cysteine residue was present between residues 551 and 775 of the alpha 1 (I) chain. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 748 of the alpha 1 (I) chain to a cysteine codon. About 80% of the type I procollagen synthesized by the proband's fibroblasts had a decreased thermal stability. The results, therefore, were consistent with the conclusion that normal pro-alpha 1 (I) chains and pro-alpha 1 (I) chains containing a cysteine residue in the alpha chain domain were synthesized in about equal amounts and incorporated randomly into type I procollagen. However, only about 10% of the alpha 1 (I) chains generated by trypsin digestion were disulfide-linked. Further studies demonstrated a decreased rate of secretion of type I procollagen containing the new cysteine residue and decreased processing of the protein by procollagen N-proteinase in cultures of postconfluent fibroblasts. Both parents were phenotypically normal and their fibroblasts synthesized only normal type I procollagen. Therefore, the mutation in the proband was a sporadic one and is very likely to have caused the connective tissue fragility that produced the lethal phenotype.     

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s)

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.     

Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains.

We have previously shown that type I procollagen pro-alpha1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995) J. Biol. Chem. 270, 8642-8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-alpha1(I), were stably transfected with a pro-alpha1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-alpha1(I) could be detected indicating that the majority of the mutant pro-alpha1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH(4)Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-alpha1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-alpha1(I) in OI26 is mediated by proteasomes.     

A type I collagen with substitution of a cysteine for glycine-748 in the alpha 1(I) chain copolymerizes with normal type I collagen and can generate fractallike structures

Type I procollagen was purified from cultured fibroblasts of a proband with a lethal variant of osteogenesis imperfecta. The protein was a mixture of normal procollagen and mutated procollagens containing a substitution of cysteine for glycine in either one pro alpha 1(I) chain or both pro alpha 1(I) chains, some or all of which were disulfide-linked through the cysteine at position alpha 1-748. The procollagen was then examined in a system for generating collagen fibrils de novo by cleavage of the pCcollagen to collagen with procollagen C-proteinase [Kadler et al. (1987) J. Biol. Chem. 262, 15696-15701]. The mutated collagens and normal collagens were found to form copolymers under a variety of experimental conditions. With two preparations of the protein that had a high content of alpha 1(I) chains disulfide-linked through the cysteine alpha 1-748, all the large structures formed had a distinctive, highly branched morphology that met one of the formal criteria for a fractal. Preparations with a lower content of disulfide-linked alpha 1(I) chains formed fibrils that were 4 times the diameter of control fibrils. The formation of copolymers was also demonstrated by the observation that the presence of mutated collagens decreased the rate of incorporation of normal collagen into fibrils. In addition, the solution-phase concentration at equilibrium of mixtures of mutated and normal collagens was 5-10-fold greater than that of normal collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human placenta type V collagens. Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule

Human type V collagen was purified from placenta and found to contain alpha 1(V), alpha 2(V), and alpha 3(V) chains in varying ratios. Using any of three independent nondenaturing methods (phosphocellulose chromatography, high-performance ion-exchange chromatography on IEX-540 DEAE, and ammonium sulfate precipitation), this preparation could be resolved into two fractions. Analysis of the two fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one fraction contained alpha 1(V) and alpha 2(V) in a 2:1 ratio and the other contained alpha 1(V), alpha 2(V), and alpha 3(V) in a 1:1:1 ratio. When the crude placental type V collagen was electrophoresed under nondenaturing conditions, two bands were observed, one co-migrating with purified (alpha 1(V]2 alpha 2(V) and the other co-migrating with the fractions containing alpha 1(V), alpha 2(V), and alpha 3(V) chains in a 1:1:1 ratio. Electrophoresis in a second dimension under denaturing conditions confirmed that the fast-migrating band contained (alpha 1(V]2 alpha 2(V) and that the slow-migrating band contained the three chains in equimolar ratio. CD spectra of the two fractions and resistance to trypsin-chymotrypsin digestion confirmed that the two fractions contain triple helical collagen. Thermal denaturations were monitored by the changes in CD signal at 221 nm. The two fractions purified by ammonium sulfate precipitation melted at 39.1 and 36.4 degrees C for the (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) fractions, respectively. Trypsin cleavage of these two native fractions at temperatures near melting produced completely different fragmentation patterns, indicating different partial unwinding sites of the alpha 1(V) and alpha 2(V) chains in the two preparations and thus different molecular assemblies. Our data demonstrate the existence of two different molecular assemblies of type V collagen in human placenta consisting of (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) heterotrimers.