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.     

Identification of collagen alpha1(I) trimer and normal type I collagen in a polyoma virus-induced mouse tumor.

A bone- and cartilage-forming mouse tumor, induced by transforming salivary epithelial cells with polyoma virus, contained large quantities of collagen. Two types of collagen molecules were isolated which had different solubilities in salt. One was type I collagen with a chain composition [α1(I)]₂ α2 and the other was an unusual form of type I collagen with a chain composition [α1(I)]₃. This would appear to be the first in vivo demonstration of α1 type I trimer.     

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.     

Characterization of notochord collagen as a cartilage-type collagen

Sturgeon notochord and cartilage collagens have been characterized with respect to chromatographic properties, amino acid composition, carbohydrate content, and cyanogen bromide cleavage products of the component α chains. The data show that the collagen of both tissues is comprised of a single type of α chain and that the notochord and cartilage chains are identical. Further, the sturgeon chains bear a striking resemblance to previously characterized α1(II) chains from avian and mammalian hyaline cartilages. These observations strongly suggest that the data may be extrapolated to higher organisms and indicate that during development, a cartilage-type collagen is synthesized by notochord cells prior to the appearance of tissues classically identified as cartilage on the basis of morphology.     

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.     

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.     

Quantitation of collagen in polyacrylamide gels by fluorescent scanning of MDPF1-labeled proteins: An improvement over densitometric scanning of gels stained by coomassie blue

Purified α chains of collagen were made fluorescent by coupling with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) and then electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. The migration of MDPF-labeled collagen was similar to unlabeled collagen α chains except that MDPF-α1(I) migrated closer to MDPF-α2. The area under the peaks recorded from fluorometric scanning of MDPF-labeled α1(I), α2, and α1(III) was linear from 10^?5 to 10^?8 g. The standard curves for the three α chains were similar. The results from nonreplicate determinations had ±6% SE. This method is an improvement over staining with Coomassie blue. It has a greater sensitivity, peak area is independent of migration distance, has a wider range of linearity, and permits observation of bands during electrophoresis with quantitation immediately after electrophoresis.     

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.     

Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.     

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

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.     

FBJ virus-induced osteosarcoma has type V collagen consisting of A, B and C-like chains in addition to type I collagen

A study was carried out on collagen chains of FBJ virus-induced osteosarcoma. Collagens were extracted from pepsin-digested tissues and fractionated by differential salt precipitation. An acidic 0.7 M NaCl precipitate contained type I, type I trimer and/or type III collagens. Collagen fractions precipitated at acidic 1.2 M NaCl showed features characteristic of type V collagen consisting of three chains (mol. weights of which were 120K, 110K and 100K daltons). None of these chains, however, was identical to any of the B, C or A chains reported by Sage et al. in 1979 (1), judging from amino acid composition, cyanogen bromide cleavage and phosphocellulose chromatography data.     

Characterization of lens capsule collagen: evidence for the presence of two unique chains in molecules derived from major basement membrane structures

A collagen fraction representing two-thirds of the collagenous sequences in bovine lens capsules has been isolated following limited pepsin digestion and purified by DEAE- and carboxymethyl-cellulose chromatography in native form. The denaturation products of this collagen contain two types of components. The more acidic components (C and 50K₁) are, respectively an α-chain-sized collagenous polypeptide and a mixture of smaller molecular weight proteolytic cleavage products of the C chain. The more basic components (80K and 50K₂) represent, respectively, a collagenous polypeptide with an apparent M_r = 80,000 and a mixture of smaller molecular weight components derived through proteolysis of the 80K component. The C chain and 80K components are unique with respect to chromatographic properties, amino acid composition, and cyanogen bromide cleavage products. These data are interpreted to indicate that lens capsule basement membrane collagen molecules collectively contain at least two genetically distinct collagen chains: the C chain representing the collagenous domain of one type of chain and the 80K component representing the major portion of the collagenous domain of a second type of chain, designated the D chain.     

In vitro cartilage formation: effects of 6-diazo-5-oxo-L-norleucine (DON) on glycosaminoglycan and collagen synthesis.

To determine the relationships between glycosaminoglycan (GAG) synthesis and type-specific collagen synthesis, we have investigated mouse limbs cultured in the presence of antiglutamine DON (6-diazo-5-oxo-l-norleucine). When compared to control limbs, ultrastructural examination of the DON-treated limbs shows that newly formed cartilage lacks matrix granules and the collagen fibrils have an altered morphology. Using [³⁵S]sulfate as a precursor, we have found that DON (5 μg/ml) suppresses chondroitin sulfate synthesis to less than 15% of the control level. We have also examined the collagen synthesized in equivalent limbs labeled with [³H]proline. The α-chain patterns from CM-cellulose chromatography were very similar for control and experimental limbs (α1:α2 ～ 7), suggesting that both (α1)₃- and (α1)₂α2-type molecules were being produced. The (α1)₃ molecules in both cases were identified as type II collagen by fractional salt separation and cyanogen bromide peptide mapping on CM-cellulose columns. We conclude that (1) the synthesis of type II collagen can be dissociated from the production of GAG, and (2) environmental influences can be involved in controlling the fibrillogenesis of collagen.     

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.     

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.     

Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels

The differentiated phenotype of rabbit articular chondrocytes consists primarily of type II collagen and cartilage-specific proteoglycan. During serial monolayer culture this phenotype is lost and replaced by a complex collagen phenotype consisting predominately of type I collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes reexpress the differentiated phenotype during suspension culture in firm gels of 0.5% low T_m agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains. The rates of proteoglycan and collagen synthesis return to those of primary chondrocytes. Using SDS-polyacrylamide gel electrophoresis of intact collagen chains and two-dimensional cyanogen bromide peptide mapping, we demonstrated a complete return to the differentiated collagen phenotype. These results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression.     

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.     

The collagen of heart valve.

A hydroxylysine-rich type I collagen has been isolated from pepsin-digested porcine heart valve. The ratio of alpha1 to alpha2 in the isolated molecule was 2:1. The component alpha chains exhibited unusual chromatographic behavior in comparison to corresponding chains from human dermis and lathyritic rat skin collagen. The composition of component cyanogen bromide peptides identified the alpha chains as authentic type I chains and demonstrated hydroxylysine enrichment throughout the length of the chain. delta6-Dehydro-5,5'dihydroxylysinonorleucine, a collagen cross-link derived from two hydroxylysyl residues and ordinarily found in hard tissue collagens was found to be the predominant cross-link in heart valve.     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).