Hydroxylysine Residues Research Articles

The effect of ascorbic acid on the cross-linking of collagen during its synthesis by cultured 3T6 fibroblasts

Since hydroxylysine has been shown to play a critical role in the intermolecular cross-linking of collagen and since the hydroxylation of lysine, like proline, is ascorbic acid dependent, it was argued that ascorbic acid deficiency should impair this type of cross-linking of collagen. This was tested in collagen newly synthesised in tissue culture by 3T6, a mouse fibroblast line. Ascorbic acid deficiency resulted in half the amount of hydroxyproline-containing material being laid down; this material was more salt soluble than the collagen from ascorbate-fed cultures, but behaved identically on disc gel electrophoresis, thus supporting the hypothesis that ascorbic acid affects the intermolecular cross-linking of collagen via certain hydroxylysine residues as well as affecting the hydroxylation of proline. Final proof must await the isolation of the cross-links.

Cellular regulation of collagen biosynthesis.

THE synthesis of collagen involves several enzymatic modifications of the polypeptide precursor of collagen, protocollagen1–4. First, certain proline and lysine residues in protocollagen are hydroxylated in similar enzymatic reactions involving molecular 02, Fe2+, α-ketoglutarate and ascorbic acid5–8. Much of the hydroxylation occurs after completed polypeptide chains have been released from the polyribosomal complexes9–11. Some hydroxylysine residues are then glycosylated by the stepwise addition of glucose and galactose, to form disaccharide appendages on the polypeptide chain12 and this glycosylation may be essential for the secretion of collagen into the extracellular matrix13,14. Although there are conflicting reports concerning the regulatory role of the hydroxylating enzymes15–20, no information is available on the genetic controls of collagen biosynthesis.

Chemical studies on Clq; A modulator of immunoglobulin biology

Summary Complete amino acid analyses of purified Clq from human serum indicate that this glycoprotein has an unusual composition for a serum protein, which includes hydroxyproline, hydroxylysine and high levels of glycine. A majority of the hydroxylysine residues are resistant to periodate oxidation; presumably they are protected by linkages to carbohydrate groups. In addition the total carbohydrate content is 7.7%, consisting primarily of glucose and galactose in equimolar amounts. Smaller quantities of sialic acid, fucose, mannose, galactosamine and glucosamine were also found. Thus, the data indicate that Clq has a structural similarity to collagen and collagen-like proteins.

Murine amyloid fibril protein: isolation, purification and characterization.

Murine amyloid has been produced by four different induction methods ( Mycobacterium butyricum, casein, casein plus Freund's adjuvant and endotoxin-induced mouse amyloidosis) in several strains and obtained from mice with "spontaneous" amyloidosis. The amyloid fibrils have been concentrated from spleen and liver. Electron microscopy of all of these preparations reveals the amyloid fibril to be 100 Å in width and composed of two parallel filaments, each measuring 35-40 Å in width and having the appearance of a twisted ribbon. X-ray diffraction of all preparations reveals a "backbone" spacing at 4.75 Å and a "side chain" spacing at 11 Å indicating a "β-pleated sheet" structure. Identification of the major protein component of amyloid fibril concentrates was made by combined use of sodium dodecyl sulfate polyacrylamide disc electrophoresis, labeling of the protein with 3H-tryptophan and Sepharose 4B gel filtration. Purification of the amyloid protein from spontaneous amyloidosis liver was accomplished by sequential gel filtration with 5 M guanidine in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns. The material is a unique glycoprotein with a molecular weight of 7200, a high content of dicarboxylic and short chain amino acids, a significant amount of tryptophan and an unreactive NH2-terminal amino acid, tentatively identified as pyrrolid-2-one-5-carboxylic acid. There are no methionine, half-cystine, hydroxylysine or hydroxyproline residues. Murine amyloid protein, therefore, has striking similarities to many human amyloid protein preparations. It differs from the human proteins in the similarity of molecular weights of different preparations.

The Lens Capsule: STUDIES ON THE CARBOHYDRATE UNITS

Abstract A study has been made of the carbohydrate units present in the anterior and posterior portions of the lens capsules of calf and cow eyes. Digestion of the capsules with purified collagenase resulted in the solubilization of more than 90% of the carbohydrate and peptide portions. Further digestion with Pronase was employed to obtain the carbohydrate units in the form of glycopeptides with a minimum number of amino acids attached. Fractionation of the collagenase-Pronase digests by gel filtration and ion exchange chromatography permitted the isolation of glycopeptides containing a glucosylgalactose disaccharide linked to hydroxylysine, glycopeptides containing heteropolysaccharide units, and some incompletely digested material believed to contain both of these types of carbohydrate units. Chemical (periodate oxidation, methylation, and graded acid hydrolysis) and enzymatic characterization of the glycopeptides containing glucose and galactose, as well as of the disaccharide isolated from them by partial acid hydrolysis, indicated that the structure of the disaccharide unit is 2-O-α-d-glucosyl-d-galactose and that it is linked by a β-glycosidic bond to the hydroxyl group of hydroxylysine. Measurement of the number of such disaccharide units was achieved on the amino acid analyzer as glucosylgalactosylhydroxylysine after alkaline hydrolysis of the lens capsules. The migration of the glucosylgalactosylhydroxylysine from the lens capsules was identical, both by ion exchange and paper chromatography, with that of the glucosylgalactosylhydroxylysine previously obtained from the gomerular basement membrane. The lens capsules were found to have a large number of hydroxylysine-linked disaccharide units (37 per 1000 amino acid residues for the calf anterior capsule and 45 per 1000 for cow anterior capsule). All of the glucose and a large portion of the galactose of the lens capsule was accounted for in the form of this disaccharide unit. Approximately 85% of the hydroxylysine residues of the capsules were involved in these carbohydrate linkages. The glycopeptides containing the heteropolysaccharide units were excluded from Sephadex G-25, penetrated Sephadex G-50, and contained galactose, mannose, hexosamines, sialic acids, and fucose. Their composition and behavior during gel filtration was similar to the glycopeptides containing the heteropolysaccharide unit from the gomerular basement membrane. The ratio, on a weight basis, of disaccharide units to heteropolysaccharide units varied with age and position in the capsule, being 69:31 for the calf anterior capsule, 63:37 for the calf posterior capsule, 85:15 for the cow anterior capsule, and 77:23 for the cow posterior capsule.

Characterization and Quantitative Determination of the Hydroxylysine-linked Carbohydrate Units of Several Collagens

Abstract The hydroxylysine-containing glycopeptides from collagenase-Pronase digests of bovine tendon collagen were found to have glucose and galactose as their monosaccharide components. The ratio of galactose to glucose in these glycopeptides was greater than unity and after alkaline hydrolysis both glucosylgalactosylhydroxylysine and galactosylhydroxylysine were found. In order to determine the content of glucosylgalactosylhydroxylysine, galactosylhydroxylysine, and unsubstituted hydroxylysine present in tendon collagen as well as in several other collagens, analyses for these components were performed on the amino acid analyzer after alkaline hydrolysis. The other collagens studied were obtained from rat skin, rat tail tendon, carp swim bladder, rabbit sclera, and corneas from rabbits and calves. From most of these sources both the citrate-soluble and citrate-insoluble collagens were studied. In all of the proteins studied both glucosylgalactosylhydroxylysine and galactosylhydroxylysine were found, with the galactosylhydroxylysine making up from 46% of the total hydroxylysine-linked carbohydrate units in calf skin citrate-soluble collagen to 11% in carp swim bladder citrate-insoluble collagen. The citrate-insoluble collagens tended to contain a greater percentage of glucosylgalactosylhydroxylysine units than the citrate-soluble collagens from the same source. The percentage of the total hydroxylysine residues substituted by the glycosidic linkages of these units varied from 5% in rat tail tendon citrate-soluble collagen (0.4 carbohydrate unit/1000 amino acid residues) to 56% in the rabbit cornea citrate-insoluble collagen (5.8 carbohydrate units/1000 amino acid residues). All these collagens were shown to contain markedly fewer hydroxylysine-linked carbohydrate units than basement membranes, suggesting that higher levels of hydroxylysine-linked carbohydrate are reflected in lower degrees of morphological organization as seen under the electron microscope. The glucosylgalactosylhydroxylysine and galactosylhydroxylysine obtained from the alkaline hydrolyses of the various collagens had the same migration on paper and ion exchange chromatography as the 2-O-α-d-glucosyl-O-β-d-galactosylhydroxylysine and O-β-d-galactosylhydroxylysine previously isolated from the bovine renal glomerular basement membrane. For structural studies glycopeptides containing the disaccharide units were obtained from the collagenase-Pronase digest of ichthyocol. The preferential release of glucose during graded acid hydrolysis of these glycopeptides and the isolation of a disaccharide with galactose on the reducing end indicated a glucosylgalactose sequence in these carbohydrate units. The disaccharide was split by α-glucosidase but was resistant to the action of β-glucosidase. Periodate oxidation, galactose oxidase treatment, and methylation of the glycopeptides indicated that the glucose is linked glycosidically to carbon-2 of the galactose. These results are consistent with a structure for the disaccharide of 2-O-α-d-glucopyranosyl-d-galactose, which is identical with that found for the glomerular basement membrane. In addition to glucose and galactose, the collagens contained small amounts of other sugar components including mannose, fucose, hexosamines, and sialic acids. In the collagenase-Pronase digests of ichthyocol and tendon collagen, glycopeptides containing these sugars could be separated from most of the hydroxylysine-containing glycopeptides by gel filtration.

Some properties of collagen modified by the Mannich reaction

AbstractThe Mannich reaction of several aliphatic active hydrogen compounds with formaldehyde and collagen, the principal protein of animal hides and skins, was investigated. These reactions were all carried out under extremely mild conditions: aqueous solutions buffered at about pH 4 and at or slightly above room temperature. The sites of the reaction in the protein were found to be the lysine, hydroxylysine, and histidine residues. The extent of the reaction and the effects of various factors on the reaction also were determined. Although polyfunctional active hydrogen compounds were used, there was little indication that cross‐linking had taken place. With acetylacetone and methyl acetoacetate the evidence indicated that dihydropyridines were formed. The extent of such a reaction was calculated from ultraviolet absorption data and from the amino acid analysis of the chemically modified proteins. The results of the two methods were in extremely good agreement with each other. With malonic acid a product was formed that offers potential for practical application. A number of new compounds were found in the hydrolyzate of the product. Two of these were identified as N ϵ‐(β‐carboxyethyl)‐L‐lysine and Nϵ, Nϵ‐bis(β‐carboxyethyl)‐L‐lysine.

Studies on the Renal Glomerular Basement Membrane: NATURE OF THE CARBOHYDRATE UNITS AND THEIR ATTACHMENT TO THE PEPTIDE PORTION

Abstract Digestion of bovine glomerular basement membranes with purified collagenase resulted in the solubilization of over 90% of the carbohydrate and peptide portions. After further digestion of the solubilized material with Pronase, the carbohydrate units, with only a few amino acid residues attached, were isolated by gel filtration and ion exchange chromatography. Characterization of the glycopeptides separated by these fractionation procedures indicated the presence in the basement membrane of two distinct types of carbohydrate units. One type is a disaccharide unit containing glucose and galactose, while the second type is a heteropolysaccharide consisting of galactose, mannose, hexosamines, sialic acids, and fucose, with an average molecular weight of 3500. The carbohydrate of the basement membrane appears to be equally distributed between these two types of units. There are approximately 10 disaccharide units for every heteropolysaccharide unit in the membrane. The carbohydrate-peptide linkage of the disaccharide unit was shown to be a glycosidic linkage involving the hydroxyl group of hydroxylysine, whereas the attachment of the heteropolysaccharide unit to the peptide most likely involves asparagine. Periodate oxidation studies indicated that approximately 70% of the hydroxylysine residues of the basement membrane are involved in the linkage of the disaccharide unit.

Evidence for the Linkage of a Disaccharide to Hydroxylysine in Tropocollagen

Abstract Soluble collagen from guinea pig skin has been subjected to sequential digestion with purified collagenase and trypsin. The small quantity of hexose present in the collagen can be detected in the digest largely as relatively basic glycopeptides. One of these glycopeptides has been obtained in pure form and accounts for at least 30% of the hexose of the original collagen. This glycopeptide has been shown to consist of a disaccharide of glucose and galactose linked O-glycosidically to the hydroxyl group of a hydroxylysine residue in a special peptide region of the collagen molecule. The amino acid composition and probable sequence in this region was shown to be -Gly-Met-Hyl(Glc, Gal)-Gly-His-Arg- A possible role for this structure in the formation of cross-links during collagen maturation is described.