Procollagen and collagen produced by a teratocarcinoma-derived cell line,TSD4: Evidence for a new molecular form of collagen

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of θ chains (150,000 daltons), pro α chains (130,000 daltons), and α chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (α1)₃, as did collagen isolated from the cell layer. Each α1 chain appears to be slightly larger than α1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type α1(X)₃, although similar to that of the α1 chain of type I collagen, [α1(I)]₂α2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [α1(I)]₃.     

The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes.

Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)]₃) to a mixture of Type I collagen (chain composition [α1(I)]₂α2) and the Type I trimer (chain composition [α1(I)]₃). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.     

Improved chromatographic purification of human and bovine type V collagen sub-molecular species and their subunit chains from conventional crude preparations Application to cell-substratum adhesion assay for human umbilical vien endothelial cell

Two human type V collagen sub-molecular species, designated [α1(V)]₂α2(V) and α1(V)α2(V)α3(V), were purified chromatographically from a commercially available preparation, in which cystine-rich collagenous contaminants were contained, with a column packed with Fractogel EMD SO₃⁻. From bovine crude preparations, the [α1(V)]₂α2(V) form free from the collagenous contaminants was purified. Type V collagen subunit chains were isolated from each type V collagen molecule by anion-exchange HPLC with a Bakerbond PEI Scout column. The highly purified human type V collagen molecules and their subunit chains were used to examine the inhibitory effect on human umbilical vein endothelial cell proliferation. It was confirmed that the α1(V) chain has inhibitory activity and it was found that the inhibitory effect of the [α1(V)]₂α2(V) form is stronger than that of the α1(V)α2(V)α3(V) form and that the α3(V) chain has no inhibitory activity.     

Temporal and spatial transitions in collagen types during embryonic chick limb development

To determine whether transitions occur in the types of collagen synthesized during embryonic chick limb development, the α chain composition of the collagens produced by whole limbs and various anatomical regions of limbs was analyzed at different stages (23–24 to 40). The tissues were incubated in the presence of ³H-proline and ³H-lysine and the α chain distribution of the purified, labeled collagens was determined by chromatography on carboxymethyl cellulose columns. We found that the stage 23–24 leg mesenchyme is producing predominantly, if not solely, an (α1)₂α2 type collagen (chain type as yet undetermined). At about stage 25–26 the limb core begins synthesizing detectable amounts of (α1)₃ collagen, which we presume to be cartilage type collagen, [α1 (II)]₃, while the outer portion of the limb largely continues to produce (α1)₂α2. The production of (α1)₃ collagen in the core progressively increases until, by stage 33 it is the only species detectable in the tibial diaphysis. Shortly thereafter (by stage 35⁺–36) (α1)₂α2 type collagen reappears in the tibial diaphysis signifying the production of bone collagen, [α1 (I)]₂α2. During the next several days of incubation, the relative proportion of (α1)₂α2 increases in the bony diaphysis while (α1)₃ remains the predominant species synthesized in the cartilaginous epiphysis.     

Collagen polymorphism: isolation and partial characterization of alpha 1(I)-trimer molecules in normal human skin.

Human skin has previously been shown to contain at least two genetically distinct types of collagen, type I and III. Here the presence of an additional form of collagen, α1(I)-trimer, is demonstrated. Skin collagen was solubilized by limited pepsin digestion and then fractionated by sequential precipitation with 1.5, 2.5, and 4.0 m NaCl at pH 7.4. The α-chain subunits of collagen were isolated by gel filtration and carboxymethylcellulose chromatography under denaturing conditions. The 1.5 and 2.5 m NaCl precipitates contained predominantly type I collagen with a chain composition of [α1(I)]₂α2. In the 1.5 m precipitate a small amount of type III collagen was also recovered. In contrast, the 4.0 m NaCl fraction consisted almost exclusively of α-chains which on the basis of cyanogen bromide peptide mapping were shown to be identical with α1(I). The amino acid composition of these chains was also similar to that of α1(I), except that hydroxylysine was increased and lysine was correspondingly decreased. The content of 3-hydroxyproline was also increased. These results suggest that the α-chains in α1(I)-trimer are the same gene products as α1 in type I collagen, but that the co-translational or post-translational hydroxylation of lysyl residues is more extensive in α1(I)-trimer. Estimation of the quantitative amounts of α1(I)-trimer indicated that this collagen accounts for less than 5% of the total collagen in adult human skin. It is speculated, however, that α1(I)-trimer collagen may play a role in the stability and tensile strength of normal human skin and other tissues, and defects in its biochemistry might be associated with diseases of connective tissue.     

The collagen of chick embryonic notochord

Notochords, isolated from 2 $\text{1}\text{2}$ day chick embryos, were cultured in the presence of ³H proline and the labeled proteins co-purified with chick skin carrier collagen. The purified material, most of which eluted from CM-cellulose as a single peak in the region of the carrier collagen α1 chain, contained 41% of the incorporated proline as hydroxyproline and from gel filtration measurements had a molecular weight of approximately 100,000 daltons. When the material was chromatographed on DEAE-cellulose with carrier α1 chains from both skin [α1 (I)] and cartilage [α1 (II)], it eluted predominantly with the cartilage chains.     

Retention of transformant specific type III collagen in dibutyryl cAMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant.

We previously reported that Kirsten sarcoma virus transformed BALB 3T3 (Ki-3T3) cell cultures contained mainly type I collagen and about 30% of another type designated by us as Y and which appears to be type III collagen, [α₁ (III)]₃. Clones of BALB 3T3 which exhibited contact-inhibition were found to contain mainly type I collagen [α₁(I)]₂α₂, and about 25% of another type (X) which was composed of three α₁ chains differing from those of type III (Hata, R. and B. Peterkofsky, 1977 Proc. Nat. Acad. Sci. (U.S.A.), 74: 2933—2937). Since dibutyryl 3′:5′ cyclic adenosine monophosphate (dbcAMP) increases collagen synthesis and alters other transformation specific properties of Ki-3T3 cells, we determined whether treatment of Ki-3T3 cells with this compound restored the normal collagen phenotype. We also analyzed the collagen of a revertant of Ki-3T3 which exhibits properties similar to those of the dbcAMP treated transformant. Procollagen labeled with radioactive proline was isolated from the medium or cells of cultures and was converted to collagen with pepsin; the collagen was analyzed by carboxymethyl cellulose (CMC) chromatography or gel electrophoresis under denaturing conditions. Ki-3T3 cells treated with 0.5 mM dbcAMP continued to accumulate type III collagen but there was an increase in the number of α₁ chains eluting from CMC columns in the same position as α₁ (I) suggesting increased accumulation of type X collagen. Although the revertant was similar to dbcAMP treated cells in that it exhibited a flattened morphology and a high relative rate of collagen synthesis, the collagen profile was similar to that of the transformant, consisting mainly of types I and III. These results indicate that accumulation of type III collagen is unaffected by dbcAMP but suggest that cAMP may be involved in the regulation of type X collagen. The failure of dbcAMP or reversion to affect the occurrence of type III collagen supports the mechanism of cell selection as a means of explaining the specific occurrence of type III collagen in sarcoma virus transformed 3T3 cells.     

Identification of collagen alpha1(I) trimer in embryonic chick tendons and calvaria

Collagen with a molecular composition [α₁(I)]₃ has been identified in acetic acid extracts from lathyritic chick embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development.     

Specific cleavage of the native type III collagen molecule with trypsin. Similarity of the cleavage products to collagenase-produced fragments and primary structure at the cleavage site.

Solutions of native Type III collagen (chain composition, [α1(III)]₃) exhibit a rapid and dramatic decrease in relative viscosity when incubated with trypsin. Cleavage products of the reaction were precipitated with ammonium sulfate and isolated in denatured form by molecular sieve chromatography. They were found to be comprised of: α1(III)-T1 (molecular weight, 71,000) derived from the NH₂-terminal portion of the Type III molecule; and α1(III)-T2 (molecular weight, 24,000) from the COOH-terminal portion of the molecule. Determination of the amino acid sequence at the NH₂-terminal portion of α1(III)-T2 as well as at the COOH-terminus of α(III)-T1 demonstrated that the products arose from specific cleavage of the type III molecule at an arginine-glycine bond corresponding to residues 780–781 in the repetitive triplet sequence of the α1(III) chain. The results suggest that the trypsin-susceptible bond in the native Type III collagen molecule resides in a region characterized by a relative lack of the normal collagen helicity.     

Quantitation of collagen types I,III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization

A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides. For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50°C, 70/60 cm×70 μm I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [α₁(I)CB₂ and α₁(I)CB₄] (constituting a fused peak) along with α₁(III)CB₂ and α₁(V)CB₁. Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20–30 runs.     

Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10–20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or α1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the α1(I) chain and to all cyanogen-bromide-treated peptides tested (α1-CB2, α1-CB3, α1-CB4, α1-CB5, α1-CB6A, α1-CB7, α1-CB8, α2-CB2, α2-CB3 and α2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited intial cell attachment, an effect that persisted longer on substrates made of α1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.     

Characterization of the collagen synthesized by cultured human smooth muscle cells from fetal and adult aorta.

Smooth muscle cells were grown from explants of the tunica media of fetal and adult human aorta. Collagen was isolated after incubation with [14C]glycine and was characterized by ion-exchange chromatography. All cells investigated synthesized two types of collagen: Type I (chain composition [alpha1(I)]2alpha2) and type III (chain composition [alpha1(III)]3). The collagen made by cells from adult donors contained approximately 70% type I and 30% type III collagen. This corresponds to the collagen composition in teh original tissue. No age-relate change in the type I/type III ratio was found with cells from donors between 9 and 67 years of age. On the other hand, the type III portion of the collagen made by fetal cells was markedly less (about 15-20% of total collagen).     

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.     

Synthesis of procollagen by odontogenic cells of rabbit tooth germ

Studies were performed to determine whether cultured odontogenic cells from rabbit tooth germ (RP cell) could synthesize dentine-like collagen. When cells were cultured with [¹⁴C]proline, 33% of the total incorporated proteins present were collagenous. Cultured RP cells were labelled with [¹⁴C]proline in the presence of β-aminopropionitrile. The resulting fractions, on analysis by CM-cellulose chromatography, contained three radioactive protein peaks, α1(I), [α1(III)]₃, α2. From the radioactive measurements, RP cells synthesized a significant amount of type III collagen, comparable to type I collagen.DEAE-cellulose chromatography was used to separate collagen molecules from collagen precursors. The results showed that 60% of total collagen precursor was type III precursor and the remainder was type I precursor.CM-cellulose chromatography of CNBr peptides of collagen from culture medium and cell extract revealed the presence of type I and type III collagen. Thus, the RP cell, which is a diploid cell, is unique in the predominance of type III collagen in culture, differing thereby from the character of collagen in vivo.     

Collagen synthesis by regenerating rabbit ear tissue

The principal collagen types synthesized during two distinct phases of regeneration in rabbit ears have been investigated, in order to relate altered phenotypic expression in connective tissue cells to regeneration of cartilage. To do this, radioactively labeled collagens synthesized in short-term culture by selected regenerating ear tissues were analyzed by ion-exchange chromatography and SDS-gel electrophoresis of the intact collagens and of the cyanogen bromide peptides derived from them. Prior to the appearance of cartilage, rabbit ear holes are filled by an outgrowth of mesenchyme-like cells derived locally from adjacent tissues. These cells produce a mixture of collagens including type I, [α1(I)]₂α2, and the type I trimer, [α1(I)]₃, but not type II collagen. Trimer production represents about one-fourth of the collagen synthesized in either a 4-, 10-, or a 24-hr incubation. Trimer is not made by tissues from healing skin wounds nor is it present in normal, uninjured ear tissues. Type II collagen synthesis was detected in tissues taken from late regenerates containing histologically recognizable cartilage, and direct analysis of regenerated cartilage confirmed the presence of type II collagen in the matrix. Thus, regenerated cartilage in the rabbit ear system contains the normal cartilage collagen, type II, while the proliferating cell mass from which the cartilage develops synthesizes the unusual collagen, [α1(I)]₃.     

Temporal and spatial transitions in collagen types during embryonic chick limb development : II. Comparison of the embryonic cartilage collagen molecule with that from adult cartilage

Analyses were performed to compare the (α1)₃ collagen molecule synthesized by the cartilaginous long bone anlage of 8-day chick embryos with that produced by adult cartilage, [α1(II)]₃. The embryonic molecule produced segment-long-spacing crystallites which had a banding pattern indistinguishable from that of authentic [α1(II)]₃. After carboxymethyl cellulose chromatography, the material in the α1 chain peak of the embryonic collagen was found to have the same amino acid composition as that of the α1(II) chain.     

Defective collagen VI α6 chain expression in the skeletal muscle of patients with collagen VI-related myopathies

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.     

Nanoscale morphology of Type I collagen is altered in the Brtl mouse model of Osteogenesis Imperfecta

Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype–phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen–ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.     

Separation of β22 dimer from bovine bone collagen

The precise role of the α₂-chain in collagen type I is of considerable scientific interest. Our recent studies demonstrated that the most noticeable difference between type I collagens, which were obtained from bovine hard tissues (bone, dentine) and soft tissues (tendon, skin), was presented in the position of β chain dimers using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The additional band observed both in the bone and dentine collagen was putatively identified as β₂₂ dimer (made of by an intermolecular cross-linking between two α₂-chains). Further investigations carried out on bovine bone and skin collagen, corresponding to hard tissue and soft tissue collagen respectively, confirmed this hypothesis. Successful separation of individual β₂₂ dimer from bone collagen was achieved. The procedure involves molecular-sieve chromatography on a Sephacryl S-400 column followed by differential acetone precipitation. Identification was done by the widely used methods, such as SDS-PAGE and cyanogen bromide (CNBr)-cleaved peptide analysis. It was proposed that the dimer and consequently α₂-chains may play important roles in the morphological and biological differences between hard and soft tissues.