Chymotryptic and tryptic peptides of fragment alpha 1-CB3 from bovine corneal collagen. Pinpointing the sites of hexose attachment.

Tryptic peptides of citraconylated fragment alpha1-CB3 and chymotryptic peptides of fragment alpha1-CB3 of bovine corneal collagen were prepared, isolated and characterized. Their amino acid compositions were consistent with the amino acid sequence of fragment alpha1-CB3 from calf skin collagen. Two glycoside sites were identified in bovine corneal fragment alpha1-CB3, one of them being the first located in the overlap region of collagen. The results are related to the uniformly narrow collagen fibres found in cornea and essential for its transparency.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

USE OF ULTRASONIC SPECTROSCOPY AND VISCOSIMETRY FOR THE CHARACTERIZATION OF CHICKEN SKIN COLLAGEN IN COMPARISON WITH COLLAGENS FROM OTHER ANIMAL TISSUES

Use of traditional sources of collagen such as pork, bovine, and carp has some limitations. Chicken skin can be valuable alternative. In this work collagen was isolated from chicken skin using a modified procedure. Molecular properties of chicken collagen were analyzed and compared to collagen from other animal skins. Acid-soluble collagen type I was obtained with a yield of 25% and water content around 67%. Viscosimetry and ultrasonic spectroscopy were newly used for molecular characterization. By ultrasonic attenuation measurements, a pre-aggregation phase in the interval from 20°C to 27°C was observed, which is a proof of disaggregation and liquefaction. From 40°C upward, the liquefaction process finishes and aggregation continues. In a bovine sample this phenomenon starts at 40°C, in chicken at 50°C, and continues until 70°C. By viscosimetry, the denaturation temperature was confirmed as 40°C for bovine and 50°C for chicken collagen. Chicken collagen has a two times higher lysine level than bovine, which provides molecular stability side-chain interactions. With regard to higher thermal stability and favorable amino acid composition, waste chicken skin has the potential to be an excellent alternative source of raw collagen with applications in the food industry and biomedicine.     

Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen

A stable nonreducible trifunctional cross-linking amino acid has been isolated from mature bovine skin collagen fibrils. Previous cross-link peptide isolations and amino acid analyses indicate the compound has properties identical with those of hydroxyaldolhistidine. Its newly proposed structure was verified using fast atom bombardment mass spectrometry, and 1H and 13C nuclear magnetic resonance. The data indicated that the cross-link consists of the prosthetic groups from one residue each of histidine, hydroxylysine, and lysine. The 1H and 13C nuclear magnetic resonance data indicated that imidazole C-2 of histidine is linked to C-6 of norleucine (epsilon-deaminated lysine residue) which in turn is linked to the C-6 amino group of hydroxylysine. Based on the trivial names for other cross-linking residues found in collagen and elastin it was given the name histidinohydroxylysinonorleucine. In vitro incubation studies for up to 24 weeks, in aqueous solution at physiological pH and ionic strength, using 6-month-old bovine embryo skin demonstrated a one-to-one stoichiometric relationship between the disappearance of the labile reducible bifunctional cross-link dehydrohydroxylysinonorleucine and the appearance of histidinohydroxylsinonorleucine. These results can partially explain the previously observed disappearance of dehydrohydroxylysinonorleucine with chronological age.     

Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from diamondback squid outer skin and partially characterized. The yields of acid-solubilized and pepsin-solubilized collagens were about 1.3 and 35.6%, respectively, on a dry weight basis. Pepsin-solubilized collagen was heterotrimer with a chain composition of ala2a3. The patterns of peptide fragments were different from that of porcine skin collagen. Denaturation temperature was 27.5 degrees C, about 10 degrees C lower than that of porcine collagen. The amino acid composition of pepsin-solubilized collagen from diamondback squid outer skin was similar to that from cuttlefish outer skin. This squid is big among squid species, and its skin is thick. It is clear that diamondback squid outer skin has a potential as an alternative source of collagen to bovine skin and bone. At present, collagen using aquatic materials such as skin (cod and a deep-sea fish) and scale (sea bream and anchovy) is the development stage in the related industries. Unless the problem of BSE infection in land animals is resolved aquatic materials as an alternative source of collagen will attract much attention in the cosmetic and medical fields.     

Pyridinoline, a non-reducible crosslink of collagen. Quantitative determination, distribution, and isolation of a crosslinked peptide

Pyridinoline is a crosslink compound isolated from bovine Achilles tendon collagen. It is a 3-hydroxypyridinium derivative with three amino and three carboxyl groups (Fujimoto, D., Akiba, K., & Nakamura, N. (1977) Biochem. Biophys. Res. Commun. 76, 1124-1129). The contents of pyridinoline in collagens from various sources were determined. The pyridinoline content of bovine Achilles tendon was 0.16 residue per 1,000 residues and that of rat Achilles tendon collagen was 0.017 residue per 1,000 residues. Besides Achilles tendon collagens, pyridinoline was found in collagens from costal cartilage, rib and femoral bone of rat. It was not found in collagens from the tail tendon and skin of rat. A crosslinked, triple-chained peptide containing pyridinoline was isolated from bovine Achilles tendon collagen after digestion with pronase. Its amino acid composition suggests that the peptide may be involved in an intermolecular crosslink among a carboxyterminal sequence, a sequence near the aminoterminus and a sequence in the helical region.     

Isolation and chemical characterization of collagen in bovine pulmonary tissues.

1. The contents of the fibrous proteins collagen and elastin in the pleural and parenchymal regions of bovine lungs were determined. The collagen content was approx. 70% (g/100g of salt-extracted defatted powder) in each tissue, and the elastin content was 28% in pleura and 13.5% in parenchyma. 2. Purification of the insoluble collagen from the pleura and parenchyma of bovine lungs by various methods was attempted. The collagen fractions isolated after incubation of the pulmonary tissues with the proteolytic enzymes collagenase ("collagenase-soluble" fraction) or pancreatic elastase ("elastase-insoluble" fraction) each contained approx. 87% of the total collagen initially present. 3. Both collagen fractions were chemically analysed for their amino acid and carbohydrate contents and were found to be similar to those of the intact interstitial collagens isolated from skin, bone and tendon. 4. The contents of the two aldimine cross-linking compounds, dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine, were determined in the bovine pulmonary collagen fractions, and were found to decrease with increasing age of the animals, and were similar to the values found in intact collagens from bone and tendon.     

Effects of singlet oxygen on the extracellular matrix protein collagen: oxidation of the collagen crosslink histidinohydroxylysinonorleucine and histidine

The reaction of singlet oxygen, a putative agent of skin photodamage, with the dermal collagen crosslink histidinohydroxylysinonorleucine (HHL) and its precursor histidine is reported. Reaction studies were performed with both purified HHL and bovine dermal tissue. We demonstrate that singlet oxygen can selectively oxidize HHL and histidine amino acid residues in dermal tissue and that intermediate oxidation products of histidine lead to new crosslink products. A novel mechanism for crosslink formation was proposed to involve nucleophilic addition to a transient imidazolone intermediate formed from singlet oxygen oxidation of the histidine imidazole moiety. The implication for such adduct formation and histidine oxidation in collagen proteins is the expression of aberrant collagen crosslinks, perturbation of the dermal collagen function, and hence an altered dermal state.     

奥尼罗非鱼胶原蛋白及其酸解液美拉德反应产物的研究

以奥尼罗非鱼鱼皮中胶原蛋白为研究对象,采用氨基酸自动分析仪和GC-MS分析了胶原蛋白的游离氨基酸组成、主要风味成分及其酸解液美拉德反应产物的风味成分.结果表明：鱼皮中胶原蛋白的提取率为6.61%,酸解后的游离氨基酸组成与一般胶原蛋白的氨基酸组成一致,其中风味氨基酸占52.57%;美拉德反应产物中含3,3-二甲基正辛烷、2,6,10-三甲基十二烷等20种风味成分.     

Affinity of placental decorin for collagen

Decorin was isolated from 7 M urea extract of bovine placental cotyledons by ion-exchange and hydrophobic chromatography. Decorin and its core protein showed a broad band at about 115 kDa and a single band at 47 kDa, respectively by SDS-PAGE. Anti-decorin core protein antiserum from pig skin was reacted with placental decorin and its core protein in western blotting. The NH2-terminal amino acid sequence of core protein from placental cotyledons was not different from that of core protein from skin and bone. Glycosaminoglycan of decorin was identified as dermatan sulfate by electrophoresis on a cellulose-acetate membrane and chondroitinase digestivity. Decorin bound to collagen in the order for type III, I, and V.     

The separation and amino acid analysis of collagen crosslinks on an extended basic ion-exchange column

The major reducible crosslinks found in collagen were separated and analyzed on an extended basic amino acid analyzer column. Reaction with ninhydrin allows the direct analysis of collagen crosslinks, including hydroxyaldol-histidine, a naturally occurring, nonreducible crosslink. In addition to known crosslinks, direct amino acid analysis of tissue hydrolysates reveals the presence of an unknown, ninhydrin-reactive component, in both NaB³H₄-reduced and unreduced collagenous tissues. Initial fractionation of hydrolysates on a Bio-Gel P-2 gel filtration column provides partial separaton of amino acids and crosslinks and enables more direct analysis of the crosslinks present in the samples, as well as detecting potential new crosslinks. The results also show that, prior to NaB³H₄ reduction, substantial amounts of known crosslinks are normally present in bovine skin and bone.     

Identification and partial purification of a large, variant form of type XII collagen.

A large, alternate form of type XII collagen has been identified in cultures of the human epidermoid cell line WISH. This form, designated XIIA, is comprised of alpha chains that are approximately 90 kDa larger than the 220-kDa alpha chain previously characterized in extracts of fetal chicken and bovine tissues. Results from both collagenase digestion and rotary shadow analysis of partially purified material show that the increase is due to a larger NC3 domain. While both the large (XIIA) and the small (XIIB) forms of type XII collagen are identified in pulse-chase radiolabeling of fetal bovine skin explant culture, they are not related in a precursor-product fashion. Inhibition studies with alpha, alpha'-dipyridyl indicate that proper folding of the collagen helix is required for complete assembly and secretion of type XIIA in WISH cell culture. The 310-kDa alpha 1A chain is likely to represent the bovine equivalent of a second translation product, estimated to be 340 kDa, predicted from analysis of one complete chick cDNA sequence. Additionally, the amino-terminal amino acid sequence of the 220-kDa bovine alpha 1B chain was determined. This sequence is very near a potential alternate splice site predicted from analysis of chicken type XII cDNA.     

A simple procedure for the purification of neutral salt soluble type I collagen from skin

Intact monomeric type I collagen was purified from fetal bovine skin by a simple and time saving procedure. Saline precipitates of mixed skin collagens, in 4 M NaCl, were extracted with a limited volume of dilute acetic acid, taken in the proportion of 1 ml per g of original wet skin; NaCl in the precipitate was not removed by dialysis. The salt concentration in the extraction medium in the above conditions, selectively solubilized type I collagen.     

Evidence for natural existence of pyridinoline crosslink in collagen

Pyridinoline is a fluorescent crosslinking amino acid isolated from collagen. Recently it was claimed that this material is an artefact produced from contaminating proteins during acid hydrolysis. However, in our hands, bovine tendon collagen could not be depleted of pyridinoline by the suggested treatments. A peptide which had the same fluorescence properties as those of pyridinoline could be isolated from enzymic digests of collagen. After acid hydrolysis, presence of pyridinoline in the peptide could be demonstrated on amino acid analysis. The composition of the peptide suggests that it originates from the specific regions of collagen molecule. These results clearly indicate the existence of pyridinoline in collagen $\text{in}\text{vivo}$.     

Affinity of Placental Decorin for Collagen

Decorin was isolated from 7 M urea extract of bovine placental cotyledons by ion-exchange and hydrophobic chromatography. Decorin and its core protein showed a broad band at about 115 kDa and a single band at 47 kDa, respectively by SDS-PAGE. Anti-decorin core protein antiserum from pig skin was reacted with placental decorin and its core protein in western blotting. The NH₂-terminal amino acid sequence of core protein from placental cotyledons was not different from that of core protein from skin and bone. Glycosaminoglycan of decorin was identified as dermatan sulfate by electrophoresis on a cellulose-acetate membrane and chondroitinase digestivity. Decorin bound to collagen in the order for type III, I, and V.     

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.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

Isolation and properties of a preparation of arterial collagen solubilized by pretreatment with alkali (author's transl)]

The acid-soluble, highly cross-linked aorta collagen, of which about 30% can be converted into a soluble form by alkali treatment, followed by extraction with aetic acid, was obtained predominantly in the form of monomeric, helical molecules, as indicated by the value for the intrinsic viscosity and its behaviour in sodium dodecylsulphate disc electrophoresis. Apart from decreased values for tyrosine (0.26%), arginine (4.4%) and aspartic acid (3.9%), the amino acid composition of the aorta collagen fraction was similar to that of the acid-soluble calf skin collagen. This finding, together with the cyanogen bromide peptide pattern, shows that the collagen extracted from the artery is predominantly type I. Treatment with alkali probably shortens the alpha1-CB6-peptide by about 45 amino acids. The collagen extracted from artery was compared with acid soluble skin collagen by sodium dodecylsulphate polyacrylamide electrophoresis. The arterial collagen showed a marked increase in the rations alpha1 to alpha2 (4:1), alpha to beta (3:1) and beta11 to beta12 (2.5:1). Compared with acid soluble skin collagen, the aorta collagen contained twice as much galactose and glucose (13.5 and 9.6 nmol/mg protein respectively), which are bound to hydroxylysine. 50% of the hydroxylysine residues are unsubstituted, 15% are present as galactosyl hydroxylysine, and 35% as glucosyl-galactosyl hydroxylysine. On the basis of its reported properties, arterial collagen obtained by the method of Fujii appears to be a suitable substrate for the study of the enzymic synthesis and enzymic degradation of hydroxylysine glycosides of native arterial collagen.     

Isolation and characterization of a fluorescent material in bovine achilles tendon collagen

A fluorescent material in bovine achilles tendon collagen was isolated and characterized by ultraviolet spectroscopy, fluorescence spectroscopy and nmr spectroscopy. The data suggest that the compound is a 3-hydroxypyridinium derivative with three amino acid side chains. The name “pyridinoline” is proposed. Pyridinoline is a novel type crosslink of collagen.     

Ehrlich chromogens, probable cross-links in elastin and collagen.

(1) Proteolytic digests of tissue elastin contain material which reacts with dimethylaminobenzaldehyde in acid solution (Ehrlich's reagent) to give a cherry-pink colour. This Ehrlich chromogen(s) [EC(s)] is similar to but not identical with EC(s) previously demonstrated in tissue collagens [Scott, Hughes & Shuttleworth (1979) Biosci. Rep. 1, 611-618]. Both ECs react with diazonium salts in acid to give coloured products. (2) Diazobenzene linked via a phenolic ester to polyacrylamide beads (Biogel P10) has been used to absorb ECs specifically and almost quantitatively from proteolytic digests. The coupled deeply coloured azo-EC-peptides were then recovered after mild alkaline cleavage from the support and purified by gel chromatography. (3) Using 15N-labelled NaNO2, the collagen azo-EC-peptides were prepared, and 15N abundance measured therein. The molar absorption coefficient of the azo-EC group was calculated (18,700) based on the assumption that each azo-EC group contained one 15N atom. (4) Collagen azo-EC-peptides contained glucose and galactose, whereas elastin azo-EC peptides did not. The amino acid patterns of the two peptides were quite different, the former being rich in polar amino acids, the latter containing much alanine. The patterns were compatible with an origin from the cross-linking regions of collagen and elastin respectively. (5) Quantitative (molar) comparisons of the azo-EC group content with amino acid, amino end-group and sugar contents, and azo-EC peptide molecular mass, suggest that a structure is present in the collagen azo-EC-peptides containing two EC groups shared between four peptide chains. Three peptide chains probably meet at each (cross-linking) EC group. (6) Based on this structure, about 15% of adult bovine skin collagen contains EC groups.