Nonisotopic evaluation of collagen in fibroblasts cultures

A method for the evaluation of collagen concentrations in the medium of fibroblasts in culture was developed. Collagen was precipitated with other proteins by addition of ethanol and hydrolyzed by 6 M HCl. The primary amino acids of the hydrolyzate were reacted with o-phthalaldehyde (OPA) and secondary amino acids (Pro, Hyp) were derivatized with 9-fluorenylmethyl-chloroformate (FMOC-Cl). The mixture was separated by isocratic HPLC on a reverse-phase column. FMOC-derivatives were detected by fluorometry, whereas OPA-derivatives were not. This method is suitable for the monitoring of collagen metabolism in fibroblast cultures exposed to various effectors.     

Quantification of hydroxyproline in small amounts of skin tissue using isocratic high performance liquid chromatography with NBD-F as fluorogenic reagent

In order to determine the collagen content of small amounts of skin tissue, we developed a new, simple and highly sensitive method of measuring the quantity of hydroxyproline (Hyp) using isocratic high performance liquid chromatography (HPLC) with a fluorogenic agent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F). The recovery rate of Hyp and reproducibility of the assay were high, and the test was sensitive enough to detect Hyp in less than 1 mg of skin tissue. This method is clinically useful for ensuring accurate diagnosis and for monitoring specific skin conditions using small human skin samples collected in biopsies.     

Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly

Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, (3S,2S)-l-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss of or reduction in the level of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism for explaining the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept, the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study of the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.     

Collagenous constituents of amniotic fluid.

The amniotic fluid (AF) was fractionated by dialysis, gel filtration and SDS/PAGE, and submitted to the assay of collagenous constituents. The collagenous character of peptides and proteins of amniotic fluid was confirmed by hydroxyproline (Hyp) assay and treatment with bacterial collagenase followed by electrophoresis and gel filtration of the digestion products. It was found that AF contains collagen degradation products but the classical method of Hyp determination described by Woessner (Arch. Biochem. Biophys., 1961, 93, 440-447) gives overestimated values due to the interference with other AF components. Fractionation of AF on Sephadex G-100 column allowed to remove the interfering material and to estimate the actual Hyp content which equals to approx. 6.2 microg/ml. About 70% of Hyp was found in low molecular dialyzable products and the rest (about 30%) appears to be a constituent of nondialyzable collagenous polypeptides of the molecular mass of about 7.9-26.3 kDa. It is suggested that such collagenous polypeptides may be the products of proteolytic conversion of collagen precursor (procollagen) into the monomeric form of this protein. No high molecular forms of collagen, corresponding to alpha-subunits, were found.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

The crystal structure of a collagen-like polypeptide with 3(S)-hydroxyproline residues in the Xaa position forms a standard 7/2 collagen triple helix

Collagen has a triple helical structure comprising strands with a repeating Xaa-Yaa-Gly sequence. L-Proline (Pro) and 4(R)-hydroxyl-L-proline (4(R)Hyp) residues are found most frequently in the Xaa and Yaa positions. However, in natural collagen, 3(S)-hydroxyl-L-proline (3(S)Hyp) occurs in the Xaa positions to varying extents and is most common in collagen types IV and V. Although 4(R)Hyp residues in the Yaa positions have been shown to be critical for the formation of a stable triple helix, the role of 3(S)Hyp residues in the Xaa position is not well understood. Indeed, recent studies have demonstrated that the presence of 3(S)Hyp in the Xaa positions of collagen-like peptides actually has a destabilizing effect relative to peptides with Pro in these locations. Whether this destabilization is reflected in a local unfolding or in other structural alterations of the collagen triple helix is unknown. Thus, to determine what effect the presence of 3(S)Hyp residues in the Xaa positions has on the overall conformation of the collagen triple helix, we determined the crystal structure of the polypeptide H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH to 1.80 A resolution. The structure shows that, despite the presence of the 3(S)Hyp residues, the peptide still adopts a typical 7/2 superhelical symmetry similar to that observed in other collagen structures. The puckering of the Xaa position 3(S)Hyp residues, which are all down (Cgamma-endo), and the varphi/psi dihedral angles of the Xaa 3(S)Hyp residues are also similar to those of typical collagen Pro Xaa residues. Thus, the presence of 3(S)Hyp in the Xaa positions does not lead to large structural alterations in the collagen triple helix.     

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the Arabian camel (Camelus dromedarius)

This study was conducted to determine the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen Hyp in various tissues (P < 0.01). Camel kidney showed a significantly high concentration of total, free, peptide-bound and protein-bound Hyp and collagen content as compared to other tissues examined (P < 0.01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0.01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0.01). These variations may result from differences in the collagen structure and/or composition in this species.     

Co-translational incorporation of trans-4-hydroxyproline into recombinant proteins in bacteria

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.     

胰蛋白酶消化法测定牛胶原蛋白三股螺旋结构的含量

本研究建立了一种测定胶原蛋白的三股螺旋结构含量的方法。该方法通过使用柱前衍生高效液相色谱(HPLC)法表征经胰蛋白酶酶解后胶原蛋白羟脯氨酸(Hyp)质量浓度的变化,进而对胶原蛋白的三股螺旋结构进行定量。探讨了不同的酶解时间(0~48h)、酶与底物的比例(1∶100、1∶50和1∶20)和温度(20、25、30、37℃)对明胶降解率的影响。获得了酶解的最佳条件——当胰蛋白酶与底物的比例为1∶50时,25℃酶解3h。使用该方法对明胶胶原蛋白混合液检测,结果表明,该方法能灵敏(RSD<10%)的测定胶原蛋白三股螺旋结构的含量。该方法不仅可用于生物组织研究领域,也可用于胶原蛋白食品、保健品和组织工程产品质量的评价。     

Isolation and characterization of insoluble collagen of dog hearts.

A procedure for isolating insoluble heart collagen has been developed. The method involves the use of defined optimal conditions of sonication that yield no thermal denaturation of the triple-helical structure nor disruption of the primary structure of the collagen molecules; this is followed by extraction of isolates with nondenaturing agents. The amino acid residues of the isolates are then reacted with dansyl chloride to allow determination of amino-terminal residues and quantification of the collagen. The method has several advantages over existing procedures: (i) There is no other method available for isolation of undenatured insoluble heart collagen in almost pure form (consists of 96% of type I collagen) and in a good yield. Sonication of tissue at or below 4 degrees C for a total of 120 s (15 s sonication repeated 8 times at 120-s intervals) yielded insoluble collagen fibers with 90% yield and a 20-fold purification as determined by the increase in Hyp content of the isolates. Extraction of these isolates with 0.6 M KCl and 1 M NaCl at 4 degrees C resulted in a 22-fold purification with 70% yield, while the classical extraction method with nondenaturing reagents yielded only 5-fold purification. (ii) There has been little study of the derivatization of an insoluble protein (collagen) with dansyl chloride. The Lys residues of collagen could be recovered as epsilon-Dns-Lys in 84% yield from a reverse-phase C-18 column by high-performance liquid chromatography. This assay allows measurement of 0.1-100 nmol epsilon-Dns-Lys. (iii) The method generates direct information concerning the quantity of collagen and its nature with respect to amino groups.     

Effect of sodium fluoride and magnesium chloride on different hydroxyproline fractions in rat liver

Sodium fluoride (NaF) is used for prevention of caries in the form of fluoridated drinking water, fluoride tablets etc. In the present study, the effect of NaF-induced alterations in hydroxyproline (Hyp) and collagen was investigated in rat liver. The effect of pretreatment with MgCl2 on NaF-induced changes in liver Hyp and collagen was also studied. The NaF treatment at 5, 10 and 20 mg/kg body wt (reported LD50 of NaF being 24 mg/kg body wt through intraperitoneal route) caused a significant decrease in free Hyp (P < 0.05), when compared to control rats. The rats treated with 20 mg/kg body wt of NaF showed a significant increase in protein-bound Hyp (P < 0.001), as compared to control group, while of NaF treatment at 5 and 10 mg/kg body wt caused no significant change in protein-bound Hyp. All the doses of NaF had no significant effect on peptide-bound and total Hyp and total collagen. Treatment of with MgCl2 alone (30 mg/kg body wt) or with NaF (10 mg/kg body wt) caused a significant decrease in free Hyp (P < 0.05). MgCl2 alone and with NaF caused a significant increase (P < 0.05) in total collagen content. Thus, the present study demonstrated that NaF had no significant effect on total Hyp and collagen, indicating that its use in various products may not interfere with the liver collagen.     

Increasing effect of an oral intake of L-hydroxyproline on the soluble collagen content of skin and collagen fragments in rat serum

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.     

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Increasing Effect of an Oral Intake of L-Hydroxyproline on the Soluble Collagen Content of Skin and Collagen Fragments in Rat Serum

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.     

Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens

Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986), except α1(III), have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl) all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in α1(III) from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707). In mammals only α2(I) and α2(V) chains had any 3Hyp at the A3 site, whereas in chicken all α-chains except α1(III) had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus α1(I). Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues.     

Effect of silver nanoparticles and hydroxyproline,administered in ovo,on the development of blood vessels and cartilage collagen structure in chicken embryos

It has been considered that concentrations of certain amino acids in the egg are not sufficient to fully support embryonic development of modern broilers. In this study we evaluated embryo growth and development with particular emphasis on one of the major components of connective tissue, collagen. Experiments were performed on Ross 308 chicken embryos from 160 fertilised eggs. Experimental solutions of silver nanoparticles (Ag), hydroxyproline solution (Hyp) and a complex of silver nanoparticles with hydroxyproline (AgHyp) were injected into albumen, and embryos were incubated until day 20. An assessment of the mass of embryo and selected organs was carried out followed by measurements of the expression of the key signalling factors’ fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Finally, an evaluation of collagen microstructure using scanning electron microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and, in particular, the complex of AgHyp significantly increased blood vessel size, cartilage collagen fibre lattice size and bundle thickness. The general conclusion from this study is that AgHyp treatment may help to build a stronger and longer lasting form of collagen fibres.     

Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro⁹⁸⁶ in A-clade chains α1(I), α1(II), and α2(V). Two partially modified sites (A2 and A3) were found at Pro⁹⁴⁴ in α1(II) and α2(V) and Pro⁷⁰⁷ in α2(I) and α2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro⁴⁷⁰ in α2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian α1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.     

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1–5 fmol/injection (S/N=3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C₁₈ column before conducting the labelling reaction. Pro–Hyp, Pro–Gly and Pro–Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5–4.8 and 1.7–5.8%, respectively. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human urine were 97.6±28.2, 2.74±1.48, 2.08±1.13, 6.71±3.34 and 2.30±1.59 nmol/mg creatinine, respectively.     

Amino-acid sequence and cell-adhesion activity of a fibril-forming collagen from the tube worm Riftia pachyptila living at deep sea hydrothermal vents.

We have determined the amino acid sequence of the alpha chain of a fibril-forming collagen from the body wall of the marine invertebrate Riftia pachyptila (vestimentifera) by Edman degradation. The pepsin-solubilized collagen chain consists of a 1011-residue triple-helical domain and short remnants of N- and C-telopeptides. The triple-helical sequence showed one imperfection of the collagen Gly-Xaa-Yaa triplet repeat structure due to a Gly-->Ala substitution. This imperfection is correlated to a prominent kink in the molecule observed by electron microscopy. No strong sequence similarity was found with the fibril-forming vertebrate collagen types I-III, V and XI except for the invariant Gly residues. However, one of the two consensus cross-linking sequences was well conserved. The Riftia collagen shared with the vertebrate collagens many post-translational modifications. About 50% of the Pro and Lys residues are found in the Yaa position and were extensively hydroxylated to 4-hydroxyproline (4Hyp) and hydroxylysine (Hyl). A few proline residues in Xaa position were partially hydroxylated to either 4Hyp or 3Hyp. Despite the low sequence similarity, Riftia collagen was a potent adhesion substrate for two human cell lines. Cell adhesion could be inhibited by antibodies against the integrin beta 1 subunit but not by RGD peptides. This biological activity is apparently conserved in fibril-forming collagens of distantly related species but does not require the two RGD sequences present in Riftia collagen.     

The crystal structure of the collagen-like polypeptide (glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)9 at 1.55 A resolution shows up-puckering of the proline ring in the Xaa position

The collagen triple helix is characterized by the repeating sequence motif Gly-Xaa-Yaa, where Xaa and Yaa are typically proline and (2S,4R)-4-hydroxyproline (4(R)Hyp), respectively. Previous analyses have revealed that H-(Pro-4(R)Hyp-Gly)(10)-OH forms a stable triple helix, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not. Several theories have been put forth to explain the importance of proline puckering and conformation in triple helix formation; however, the details of how they affect triple helix stability are unknown. Underscoring this, we recently demonstrated that the polypeptide Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forms a triple helix that is more stable than Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). Here we report crystal the structure of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide at 1.55 A resolution. The puckering of the Yaa position 4(R)Hyp in this structure is up (Cgamma exo), as has been found in other collagen peptide structures. Notably, however, the 4(R)Hyp in the Xaa position also takes the up pucker, which is distinct from all other collagen structures. Regardless of the notable difference in the Xaa proline puckering, our structure still adopts a 7/2 superhelical symmetry similar to that observed in other collagen structures. Thus, the basis for the observed differences in the thermodynamic data of the triple helix<--> coil transition between our peptide and other triple helical peptides likely results from contributions from the unfolded state. Indeed, the unfolded state of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide seems to be stabilized by a preformed polyproline II helix in each strand, which could be explained by the presence of a unique repeating intra-strand water-mediated bridge observed in the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH structure, as well as a higher amount of trans peptide bonds.