Different pattern of collagen cross-links in two sclerotic skin diseases: lipodermatosclerosis and circumscribed scleroderma.

Changes in the process of cross-linking of collagen molecules are associated with defects in the biomechanical stability of the extracellular matrix. Fibrosis of skin is characterized by an increase in pyridinolines, which are hydroxylysine aldehyde derived cross-links usually absent in healthy skin. In this study, we analyzed cross-links in lipodermatosclerosis and localized scleroderma to address the question whether all the mature cross-links currently characterized are increased in fibrosis in addition to the increase in pyridinolines. As psoralen plus ultraviolet A treatment leads to clinical improvement of fibrotic plaques in localized scleroderma we analyzed the cross-link content in lesional skin after bath psoralen plus ultraviolet A therapy. In skin from patients with localized scleroderma an increase in the total number of mature cross-links was found to be due to an increase in both pyridinolines and dehydro-histidinohydroxymerodesmosine. The concentration of histidinohydroxylysinonorleucine was unchanged. By contrast, the total number of mature cross-links was decreased in lipodermatosclerosis. This decrease was caused by a decrease of lysine aldehyde derived cross-links (dehydro-histidinohydroxymerodesmosine and histidinohydroxylysinonorleucine), whereas the concentration of pyridinolines increased. A decrease in the content of pyridinolines after bath psoralen plus ultraviolet A treatment was found in six out of nine patients with localized scleroderma, which might reflect a remodeling of the extracellular matrix. Our data provide evidence that sclerosis of skin is associated with either an increase in the number of cross-links per molecule of collagen or a change in the molecular nature of the cross-links formed.

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.

Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin.

In fibrotic skin of lipodermatosclerosis a substantial increase of the cross-link hydroxylysylpyridinoline is observed. Hydroxylysylpyridinoline is a typical cross-link of skeletal tissue and is thought to play a major part in the hardening of sclerotic tissue. We investigated whether the increase in hydroxylysylpyridinoline is due to overhydroxylation of lysyl residues in the collagen molecule, which may also be associated with an increase of glycosylated hydroxylysine residues. Furthermore, we determined whether the collagen fibrils in lipodermatosclerosis showed a decrease of the diameter in the tissue as well as in vitro after fibrillogenesis of pepsin-solubilized collagens. Isolated alpha-chains of pepsin solubilized collagen I showed an increase in lysyl hydroxylation (hyl/(hyl + lys)) as compared with normal control [alpha1(I): lipodermatosclerosis 0.18 +/- 0.01; control 0.12 +/- 0.01; alpha2(I): lipodermatosclerosis 0.36 +/- 0.02; control 0. 25 +/- 0.03, p < 0.001]. Furthermore, the content of enzymatic glycosylated hydroxlysine residues increased. This increase is associated with a decrease of fibril diameter of both tissue and fibrils formed in vitro of pepsin-solubilized collagens. In the same pool of collagens an increase in collagen III content was observed as compared with controls (lipodermatosclerosis 14.5% +/- 1.6, control 10.3% +/- 1.6, p < 0.001). Our results showed that the overhydroxylation of lysyl residues, which is required for the generation of hydroxylysylpyridinoline, is not only restricted to the telopeptides but also affects the helical part of the molecule. This process is further associated with an increase of glycosylated hydroxylysyl residues. These changes along with the increase in collagen III content seem to be responsible for the observed alteration in the architecture of collagen fibrils in sclerotic skin.

Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro.

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.

Expression and characterization of recombinant human type II collagens with low and high contents of hydroxylysine and its glycosylated forms.

Insect cells coinfected with two baculoviruses, one coding for the pro alpha chains of human type II procollagen and the other for both the alpha and beta subunits of human prolyl 4-hydroxylase, produced the cartilage-specific type II collagen with a stable triple helix. The highest expression levels, up to 50 mg/l of type II collagen, were obtained in suspension culture using a modified construct in which sequences coding for the signal peptide and N propeptide of type II procollagen had been replaced by those for type III procollagen. The type III N propeptide artificially generated into type II procollagen was found to be cleaved at a much higher rate than the wild-type type II N propeptide, probably because the former interacted poorly with the triple-helical domain of type II procollagen. The amino acid composition of the recombinant type II collagen was very similar to that of the non-recombinant protein, but the hydroxylysine content was only 17% and that of glycosylated hydroxylysines was equally low. The hydroxylysine content was increased to the level found in the non-recombinant collagen by using an additional baculovirus coding for lysyl hydroxylase, and a substantial increase was also found in the glycosylated hydroxylysine content. No difference in thermal stability was found between the low- and high-hydroxylysine collagens.

Expression of wild-type and modified proalpha chains of human type I procollagen in insect cells leads to the formation of stable [alpha1(I)]2alpha2(I) collagen heterotrimers and [alpha1(I)]3 homotrimers but not [alpha2(I)]3 homotrimers.

Insect cells coinfected with a baculovirus coding for the proalpha1(I) chain of human type I procollagen and a double promoter virus coding for the alpha and beta subunits of human prolyl 4-hydroxylase produced homotrimeric [proalpha1(I)]3 procollagen molecules. The use of an additional virus coding for the proalpha2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proalpha1 to proalpha2 chains of type I procollagen (proalpha1(I) and proalpha2(I) chains, respectively), unless the proalpha1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proalpha1(I) chain with those of the proalpha1(III) chain increased level of expression of the proalpha1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proalpha2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. The Tm of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42-43 degrees C when determined by circular dichroism. The wild-type proalpha2(I) chain formed no homotrimers. Replacement of the C propeptide of the proalpha2(I) chain with that of the proalpha1(I) chain or proalpha1 chain of type III procollagen (proalpha1(III) chain) led to the formation of homotrimers, but the alpha2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 degrees C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proalpha2(I)]3 molecules in insect cells.

Chondronectin: physical and chemical properties.

Chondronectin, the chondrocyte attachment factor, was purified from chicken serum and characterized as to its physical and chemical properties. From sedimentation equilibrium data it was found to have a native molecular weight of 175,800 +/- 800 and a subunit molecular weight of 55,540 +/- 800 in the presence of guanidinium chloride and cysteine, suggesting a trimeric structure linked by disulfide bonds. As visualized by electron microscopy after rotary shadowing, the protein appears compact and globular. The amino acid and carbohydrate compositions of chondronectin are distinct from fibronectin, the fibroblast attachment factor, and laminin, the epithelial cell attachment factor. The activity of chondronectin in promoting attachment of chondrocytes is stable to digestion by collagenase, elastase, and neuraminidase, but is destroyed by trypsin treatment. The data suggest that chondronectin is structurally and chemically distinct from fibronectin and laminin.

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.

Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.

Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8.

The complete amino acid sequence of the 279-residue CNBr peptide CB8 from the alpha 1 chain of type I calf skin collagen is presented. It was determined by sequencing overlapping fragments of CB8 produced by Staphylococcus aureus V8 proteinase, trypsin, Endoproteinase Arg-C and hydroxylamine. Tryptic cleavages were also made specific for lysine by blocking arginine residues with cyclohexane-1,2-dione. This completes the amino acid sequence analysis of the 1054-residues-long alpha (I) chain of calf skin collagen.

Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides.

Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of human pro alpha 1 (I) and pro alpha 2 (I) procollagen chains by reverse phase high performance liquid chromatography.

Human pro alpha 1 (I) and pro alpha 2 (I) procollagen chains have been separated on a large por (33 nm) reverse phase high performance liquid chromatography C18 column using an acetonitrile aqueous gradient containing 9 mM trifluoroacetic acid. The separation of the chains was evaluated by gel electrophoresis. The yield of the chromatography, based on radiolabel recovery, is equal to or better than 80%. Absorbance monitoring at 210 nm permits detection of less than 5 micrograms procollagen.

A comprehensive approach to the study of collagen primary structure based on high-performance liquid chromatography.

A comprehensive approach for the structural microanalysis of collagen based of collagen based on high-performance liquid chromatography (h.p.l.c.) has been developed using calf skin type I collagen as a model system. The alpha, beta and gamma components were separated, after heat denaturation, on a TSK 4000 SW gel permeation column, using a nonvolatile buffer. Monitoring at 210 nm permits the detection of 1 microgram of a single chain. The alpha 1(I) and alpha 2(I) chains were completely resolved using a large-pore reversed-phase column (Vydac 201 TP 4.6) eluted by an aqueous acetonitrile gradient (24-48%) containing 0.01 M heptafluorabutyric acid as an ion-pairing agent. The purified alpha 1(I) chain was digested with CNBr and the resulting fragments separated in the same chromatography system with a gradient containing a 12.8-44.8% acetonitrile gradient. The purified alpha 1(I)CB 3 peptide was further cleaved with trypsin and the resulting peptides separated first by a similar chromatography with a 4-32% acetonitrile gradient. Resolution of some poorly separated peptides was obtained by a rechromatography using trifluoroacetic acid as counterion. The isolated peptides were hydrolyzed and identified by their amino-acid composition. Sequencing of h.p.l.c.-purified alpha 1(I)CB 3 was also performed to demonstrate the suitability of the technique for the preparation of peptides for amino-acid sequencing. This study demonstrates that detailed structural analysis can be performed on 3 mg of a purified collagen.

N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

Comparison of the NH2-Terminal sequences of chick Type I preprocollagen chains synthesized in an nRNA-dependent reticulocyte lysate.

RNA was extracted from 17-day-old chick embryo calvaria and translated by an mRNA-dependent reticulocyte lysate. Procedures were developed for purifying intact pro alpha 1(I) and pro alpha 2 translation products from the lysate. These products were identified by comparing tryptic peptide elution patterns of the translation products with those obtained from secreted pro alpha chains. Partial sequence data from the amino terminus of each translation product demonstrated that each chain begins with a sequence that is different from that of the corresponding pro alpha chain, and that the two sequences are not the same. Also, the bacterial collagenase-resistant peptide from the amino terminus of prepro alpha 1(I) had an apparent molecular weight which was 10 000 greater than the peptide from pro alpha 1(I).

Purification and characterization of the amino-terminal propeptide of Pro alpha 1(I) chains from embryonic chick tendon procollagen.

A peptide with an apparent molecular weight of 23,000 was isolated from the medium of cultured chick embryo tendons. Comparison of tryptic peptides derived from the medium peptide and from the amino-terminal, bacterial collagenase resistant portion of Type I procollagen indicated that the medium peptide represented the amino-terminal precursor-specific region of the pro alpha 1 (I) chain of procollagen. This conclusion was supported by the demonstration that antibodies against the medium peptide reacted with Type I procollagen in a radioimmune assay but did not react with a peptide derived from the carboxy-terminal propeptide of Type I procollagen. In addition, the reaction with Type I procollagen was inhibited with the purified amino-terminal, collagenase-resistant portion of pro alpha 1 (I) chains. Finally, amino acid sequencing demonstrated that the amino propeptide of dermatosparactic calf pN alpha 1 (I) chains and the medium peptide have similar amino-terminal sequences. Carbohydrate analysis established the presence of one residue of N-acetylglucosamine and a trace of mannose and galactose.