CNBr Peptide Patterns Research Articles

Isolation of noncollagenous proteins from gingival connective tissue.

Noncollagenous proteins form an integral part of gingiva and other connective tissues. We have performed studies aimed at purification and partial characterization of the gingival noncollagenous proteins. Healthy gingival tissues from mongrel dogs were extracted in neutral buffers, acetic acid, and 6 mol/L urea. Immunoblots using anti-keratin antibodies and CNBr peptide patterns revealed that the majority of the proteins present in these extracts were keratins. To exclude keratins, gingival connective tissue was separated from the epithelium and then extracted. Acid extracts of the connective tissue contained very little protein, whereas urea extracts contained collagen and other noncollagenous proteins. The noncollagenous proteins present in the urea extract were partially purified by DEAE-cellulose chromatography and separated by affinity chromatography through a Sepharose 4B-type I collagen column. At least eight proteins, which ranged in molecular size from 15 to 75 kilodaltons, were obtained by this procedure. We conclude that keratins are major components of whole gingiva extracts and that epithelium must first be removed in order for connective tissue proteins to be obtained. The gingival connective tissue appears to contain several collagen-binding proteins, and these proteins may play an important role in the structure and function of the gingival matrix.

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of α 1(IV) 95K and α 2(IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.

Separation and characterization of two polypeptide chains from the 7S cross-linking domain of basement-membrane (type IV) collagen.

The long-form 7S domain of human placental type IV collagen was prepared and after reduction, denaturation and aminoethylation, was separated into its subunits. The monomer subunit was further separated into two polypeptide chains of Mr about 25 000. From compositional data and CNBr peptide patterns it was shown that the two chains were different. Furthermore, all subunits contained both chains, thus supporting a proposed subunit structure for the 7S domain and a chain composition [alpha 1(IV)]2 alpha 2(IV) for the type IV molecule.

All three chains of 1α2α3α collagen from hyaline cartilage resist human collagenase

A previous report that the 3α collagen chain of hyaline cartilage was cleaved by human collagenase could not be confirmed when the 1α2α3α collagen fraction was freed of all contaminating type II collagen. All three minor collagen chains, 1α, 2α and 3α, were totally resistant to highly purified collagenases from both rheumatoid synovial and gastric mucosal tissues. This finding and CNBr-peptide patterns suggest that, despite the close homology with α1(II), the 3α chain is a unique collagen component, possibly combined with 1α and 2α in heterotrimeric molecules. In contrast, a 3α-like component from fibrocartilage was cleaved by collagenase and gave a CNBr-peptide pattern more typical of α1(II) than of the collagenase-resistant 3α of hyaline cartilage.

Biosynthesis and regulation of type V collagen in diploid human fibroblasts.

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival fibroblasts. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at 15 degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, 157,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival fibroblasts synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived growth factor. Platelet-derived growth factor was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived growth factor may be an important regulatory factor in the synthesis of types V and III collagens.

Altered expression of collagen phenotype in osteoarthrosis

Immunofluorescence studies have shown the presence of collagen type III in addition to types I and II in osteoarthrotic cartilage. The presence of collagen type III was verified biochemically: collagen was isolated and purified from the pronase digest of osteoarthrotic cartilage. Mixtures of different collagen types were fractionated first by two different DEAE-cellulose runs and then the collagen polymers were isolated by molecular sieve chromatography. Afterwards the collagenous material was reduced, alkylated, and rechromatographed on an agarose column. Three major peaks corresponding to -, β- and α-chains were observed. Amino acid analyses and the CNBr peptide pattern indicated the identity of the alpha peak as α 1 (III). Additional staining of control and diseased cartilages for fibronectin revealed the presence of this protein in the diseased tissue.

Two genetically distinct molecular species of octopus muscle collagen

The arm muscular tissue of an octopus, Octopus vulgaris, was subjected to limited proteolysis with pepsin and the solubilized tissue collagen was separated into two fractions by selective salt precipitation. Biochemical characterization of the major collagen fraction precipitating at 0.45 M NaCl (pH 2.6) has demonstrated that it comprises almost a single type of molecular species with structure (α1) 2α2, which was identified by its CNBr-peptide pattern as being similar to Type I-like collagen of octopus skin. On the other hand, the minor collagen fraction precipitating at 0.90 M NaCl (pH 2.6) was found to contain a unique γ-chain-sized component cross-linked by disulfide bonds. Sepharose CL-4B gel filtration of the reduced γ component revealed the presence of an α component, which is virtually identical in amino acid composition to the original γ protein and bears a close resemblance to known basement membrane collagens. This unique α component was genetically distinct from α1 and α2 chains of the major muscle collagen as judged by their CNBr-peptide patterns.

Structural differences between heavy chains of secreted and membrane-bound IgM of a human lymphoblastoid cell line

The human lymphoblastoid cell line BL was shown to synthesise three distinct molecular species of immunoglobulin M heavy chains: membrane-bound (μ m). intracellular (μ i) and secreted (μ s) μ-chains. Only the membrane-bound form could be labeled with a lipophilic photoactivatable nitrene reagent. Analysis of their constituent CNBr fragments, and carboxypeptidase A and B digestions of their C-terminal tails suggest that the CNBr peptide pattern of μ s and μ m, though similar, is not identical, and that amino acids released at the C-termini of the chains are different. The data confirm recent observations in human and murine systems by showing that the membrane-associated human μ-chain contains a hydrophobic segment, consistent with its anchorage into the lipid bilayer of the plasma membrane and a C-terminal amino acid sequence different from that of the secretory μ-chain.

Biosynthesis of two forms of IgM heavy chains by normal mouse B lymphocytes. Membrane and secretory IgM.

A study of the biosynthesis of IgM by purified mouse spleen lymphocytes showed that these cells synthesize both 8 S membrane IgM and 19S secretory IgM, which is identical with plasma cell IgM except in its kinetics of processing, assembly, and secretion. The heavy (mu) chains of these two types of lymphocyte IgM differ in their ultimate fate, in processing, isoelectric point, and peptide composition. The separate precursors of the two mu chains have very similar mobilities in sodium dodecyl sulfate polyacrylamide gel electrophoresis, but they can be distinguished by the use of endoglucosaminidase H (endo-H) to remove core sugars, by two-dimensional electrophoresis, and by one-dimensional gel analysis in pulse-chase experiments. CNBr peptide patterns of intracellular "secretory" mu chains of lymphocytes and plasma cells were similar, but membrane mu chains had a COOH-terminal peptide different in structure from that of secreted mu chains, with a higher apparent molecular weight.

Two-dimensional CNBr peptide patterns of collagen types I, II and III

CNBr peptides from the α1-chains of collagen types I, II and III have been fractionated by a high resolution two-dimensional mapping procedure. Radioactive collagen chains were synthesized without deamination of lysyl residues by rabbit cells in culture and were treated with pepsin and purified prior to cleavage by CNBr. The resultant peptides from each collagen type were separately fractionated by a two-dimensional mapping procedure consisting of non-equilibrium isoelectric focusing in the first dimension and sodium dodecyl sulfate electrophoresis in the second dimension. Unique peptide maps were obtained for each type of α-chain and only small amounts of sample were required. This method will be useful for unambiquous identification of isolated collagen chains, identification of collagens in complex mixtures, and the rapid identification of crosslinked peptides.

Characterization of collagens of diseased human gingiva.

In the gingiva and other connective tissues, alteration in the collagens is primarily responsible for their functional impairment during disease. To study the collagen alterations, we extracted diseased human gingival tissue with neutral and acidic solvents and then with pepsin. The pepsin extract was separated into proteins soluble in 2.5 and 1.5 M NaCl and proteins insoluble in 1.5 M NaCl. By the criteria of solubility behavior in NaCl solutions, elution from (carboxymethyl)cellulose (CM-cellulose) columns, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, CNBr peptide pattern, and amino acid composition, the collagens of acidic and neutral solvent extracts and 1.5 M soluble fraction of pepsin extract were characterized as type I collagen and the 1.5 M NaCl insoluble collagen as type III. The 2.5 M NaCl fraction contained alpha 2, A, and B chains. The alpha 1 chains resembled alpha 1[I] in amino acid composition, and, since alpha 2 chains were lacking, it appeared that these chains derived from type I trimer collagen. The A and B chains were purified from the 2.5 M NaCl fractions by salting out at acidic pH. The final (A plus B) chain fraction was resolved into two major and one minor protein peaks by phosphocellulose chromatography. The major peaks were characterized as A and B chains on the basis of amino acid composition and CNBr peptide patterns. The minor peak had electrophoretic mobility slightly less than B chains, and the amino acid composition was different. Analysis of the proportion of different collagen types extracted indicated that type III collagen, which is the second major fraction in other connective tissues, is only a minor constituent in the gingiva. More interestingly, A and B chains accounted for a greater proportion than type III. Unlike the fibroblast cultures, the type I trimer formed only a small proportion of collagens of diseased gingival tissue.

Biochemical characterization of collagens synthesized by intestinal epithelial cell cultures.

Rat intestinal epithelial cells in culture synthesize and secrete several different collagens as identified by biochemical and ultrastructural criteria. These collagens were labeled with [U-14C]proline, separated by DEAE- and CM-cellulose chromatography, and at least four different collagen chains were identified. Two of them, present almost exclusively as fully processed collagens, were alpha 1(I) and alpha 2 chains. The presence of a large excess of alpha 1 (I) chains over the normal ratio for type I collagen indicated that both type I collagen [alpha 1(I)]2 alpha 2 and [alpha 1(I)]3 or type I trimer were present both in the culture medium and in the extracellular matrix. The labeled alpha 1(I) chain had a CNBr peptide pattern superimposable on that of a standard alpha 1(I) chain from rat skin, but the intact radiolabeled chain differed from the standard in its migration on sodium dodecyl sulfate gels and elution position on CM-cellulose column and had a much higher hydroxylysine content. The other two collagen chains were present in the culture medium exclusively as partially processed procollagens. They constituted 60 to 70% of the collagenous proteins and had features corresponding to none of the known collagen types. They were separated by CM-cellulose chromatography and further characterized. Both contained interchain disulfide bonds in the pepsin-resistant portion of the molecule and, after pepsin digestion, eluted from CM-cellulose at lower salt concentrations than standard alpha 1(I) chain. One of them was characterized by a low hydroxyproline content and a high hydroxylysine content, with all the hydroxylysine present as glycosylgalactosyl derivative. The CNBr peptide patterns of these two unknown chains differed from each other, and did not correspond to those of any standard collagen chain examined. They may represent new collagen types specifically present in the intestinal basement membrane.

Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type

Chicken neural retina cells from 6- to 7-day embryos were labeled with [(3)H]proline for 24 and 72 hr and the collagenous proteins of the medium were analyzed. Ninety percent of the collagenous proteins eluted from DEAE-cellulose columns as a peak near the middle of the gradient. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, this material consisted predominantly of a slowly migrating procollagen, a smaller amount of an intermediate migrating form, and some alpha-chain material. Limited enzymatic digestion of this DEAE peak material plus mapping of its CNBr peptides identified this material as a type II precursor. The remaining 10% of the collagenous proteins eluted from DEAE-cellulose at the end of the gradient. Electrophoresis of this fraction showed four major bands. Two migrated near the type II precursors, a third migrated somewhat more slowly, and the fourth was near beta-chain dimers. In addition, there were two minor bands. Limited pepsin digestion of this DEAE peak material produced two bands: one migrated slightly more slowly than alpha1, and the second, considerably more slowly. The CNBr peptide pattern of this material appears to be different from any previously described. Thus, neural retina cells in culture synthesize at least two genetically distinct classes of collagenous proteins. One represents precursors to type II. The second is composed of multiple, very high molecular weight forms which may represent precursors (procollagens) of a new genetic type(s) of collagen.

Purification and characterization of proteins, including procollagen type III, in salt extracts of fetal bovine skin

The nature of high-molecular-weight proteins in salt extracts of fetal bovine skin was investigated. A series of DEAE cellulose ion-exchange columns separated the mature collagen from the high molecular weight proteins and also separated the high molecular weight proteins from each other. The following proteins were isolated: (a) a very high molecular weight protein which appears to be aggregated mature collagen; (b) two high molecular weight proteins of slightly faster mobility on SDS polyacrylamide gels, one of which is collagen-like and one of which is not; and (c) a type III procollagen, purer than those previously reported in the literature. These latter three proteins were characterized by amino acid analysis, SDS polyacrylamide gel electrophoretic mobility, collagenase sensitivity, and CNBr peptide patterns from SDS-PAGE.