Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen

Abstract Type I collagen is the most abundant collagen type in soft tissues and the only type found in mineralized bone. We established a rapid equilibrium radioimmunoassay for the carboxyterminal propeptide of human type I procollagen (PICP), to be used as an indicator of the synthesis of type I collagen. We isolated type I procollagen from the medium of primary cultures of human skin fibroblasts, digested the protein with highly purified bacterial collagenase, and purified PICP by lectin-affinity chromatography, gel filtration, and ion-exchange separation on HPLC. The purity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N-terminal amino acid sequencing of its component chains. The final radioimmunoassay was established with polyclonal rabbit antibodies. Material antigenically related to PICP is readily detected in human serum. There is only one form of the serum antigen, its molecular size and affinity to the antibodies being similar to those of the isolated propeptide. Intra- and interassay CVs are 3% and 5%, respectively. Preliminary reference intervals for healthy adults (18 to 61 years of age) are 38-202 micrograms/L for men and 50-170 micrograms/L for women: in men the concentration is inversely related to age. The serum antigen is stable during storage and after repeated thawing.

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Autophagy facilitates type I collagen synthesis in periodontal ligament cells

Scientific Reports ◽

10.1038/s41598-020-80275-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tomomi Nakamura ◽

Motozo Yamashita ◽

Kuniko Ikegami ◽

Mio Suzuki ◽

Manabu Yanagita ◽

...

Keyword(s):

Periodontal Ligament ◽

Collagen Synthesis ◽

Type I Collagen ◽

Periodontal Diseases ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Physiological Role ◽

Type I ◽

Periodontal Tissue ◽

Pdl Cells

AbstractAutophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

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Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation

Clinical Chemistry ◽

10.1093/clinchem/39.4.635 ◽

1993 ◽

Vol 39 (4) ◽

pp. 635-640 ◽

Cited By ~ 393

Author(s):

J Risteli ◽

I Elomaa ◽

S Niemi ◽

A Novamo ◽

L Risteli

Keyword(s):

Type I Collagen ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bone Collagen ◽

Collagen Molecule ◽

Type I ◽

Serum Samples ◽

Amino Terminal ◽

Wide Range ◽

Carboxy Terminal

Abstract We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

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Comparison of the Structural Characteristics of Native Collagen Fibrils Derived from Bovine Tendons Using Two Different Methods: Modified Acid-Solubilized and Pepsin-Aided Extraction

Materials ◽

10.3390/ma13020358 ◽

2020 ◽

Vol 13 (2) ◽

pp. 358 ◽

Cited By ~ 2

Author(s):

Haiyan Ju ◽

Xiuying Liu ◽

Gang Zhang ◽

Dezheng Liu ◽

Yongsheng Yang

Keyword(s):

Type I Collagen ◽

Structural Characteristics ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Dry Weight ◽

Collagen Fibrils ◽

Type I ◽

X Ray Diffraction ◽

Aggregation Structure ◽

Native Collagen

Native collagen fibrils (CF) were successfully extracted from bovine tendons using two different methods: modified acid-solubilized extraction for A-CF and pepsin-aided method for P-CF. The yields of A-CF and P-CF were up to 64.91% (±1.07% SD) and 56.78% (±1.22% SD) (dry weight basis), respectively. The analyses of both amino acid composition and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that A-CF and P-CF were type I collagen fibrils. Both A-CF and P-CF retained the intact crystallinity and integrity of type I collagen’s natural structure by FTIR spectra, circular dichroism spectroscopy (CD) and X-ray diffraction detection. The aggregation structures of A-CF and P-CF were displayed by UV–Vis. However, A-CF showed more intact aggregation structure than P-CF. Microstructure and D-periodicities of A-CF and P-CF were observed (SEM and TEM). The diameters of A-CF and P-CF are about 386 and 282 nm, respectively. Although both A-CF and P-CF were theoretically concordant with the Schmitt hypothesis, A-CF was of evener thickness and higher integrity in terms of aggregation structure than P-CF. Modified acid-solubilized method provides a potential non-enzyme alternative to extract native collagen fibrils with uniform thickness and integral aggregation structure.

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Comprehensive Assessment of Nile Tilapia Skin (Oreochromis niloticus) Collagen Hydrogels for Wound Dressings

Marine Drugs ◽

10.3390/md18040178 ◽

2020 ◽

Vol 18 (4) ◽

pp. 178 ◽

Cited By ~ 5

Author(s):

Baosheng Ge ◽

Haonan Wang ◽

Jie Li ◽

Hengheng Liu ◽

Yonghao Yin ◽

...

Keyword(s):

Wound Dressing ◽

Type I Collagen ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Wound Dressings ◽

Type I ◽

Rheological Characterization ◽

Scanning Calorimetry ◽

Soluble Collagen ◽

Collagen Hydrogels

Collagen plays an important role in the formation of extracellular matrix (ECM) and development/migration of cells and tissues. Here we report the preparation of collagen and collagen hydrogel from the skin of tilapia and an evaluation of their potential as a wound dressing for the treatment of refractory wounds. The acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted and characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), differential scanning calorimetry (DSC), circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) analysis. Both ASC and PSC belong to type I collagen and have a complete triple helix structure, but PSC shows lower molecular weight and thermal stability, and has the inherent low antigenicity. Therefore, PSC was selected to prepare biomedical hydrogels using its self-aggregating properties. Rheological characterization showed that the mechanical strength of the hydrogels increased as the PSC content increased. Scanning electron microscope (SEM) analysis indicated that hydrogels could form a regular network structure at a suitable PSC content. Cytotoxicity experiments confirmed that hydrogels with different PSC content showed no significant toxicity to fibroblasts. Skin repair experiments and pathological analysis showed that the collagen hydrogels wound dressing could significantly accelerate the healing of deep second-degree burn wounds and the generation of new skin appendages, which can be used for treatment of various refractory wounds.

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Preparation and Characterization of Thermally Stable Collagens from the Scales of Lizardfish (Synodus macrops)

Marine Drugs ◽

10.3390/md19110597 ◽

2021 ◽

Vol 19 (11) ◽

pp. 597

Author(s):

Junde Chen ◽

Guangyu Wang ◽

Yushuang Li

Keyword(s):

Type I Collagen ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Type I ◽

Salting Out ◽

Soluble Collagen ◽

Acid Soluble Collagen ◽

Skin Collagen ◽

Rat Tail

Marine collagen is gaining vast interest because of its high biocompatibility and lack of religious and social restrictions compared with collagen from terrestrial sources. In this study, lizardfish (Synodus macrops) scales were used to isolate acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). Both ASC and PSC were identified as type I collagen with intact triple-helix structures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopy. The ASC and PSC had high amino acids of 237 residues/1000 residues and 236 residues/1000 residues, respectively. Thus, the maximum transition temperature (Tmax) of ASC (43.2 °C) was higher than that of PSC (42.5 °C). Interestingly, the Tmax of both ASC and PSC was higher than that of rat tail collagen (39.4 °C) and calf skin collagen (35.0 °C), the terrestrial collagen. Solubility tests showed that both ASC and PSC exhibited high solubility in the acidic pH ranges. ASC was less susceptible to the “salting out” effect compared with PSC. Both collagen types were nontoxic to HaCaT and MC3T3-E1 cells, and ASC was associated with a higher cell viability than PSC. These results indicated that ASC from lizardfish scales could be an alternative to terrestrial sources of collagen, with potential for biomedical applications.

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Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content

Biochemical Journal ◽

10.1042/bj1890111 ◽

1980 ◽

Vol 189 (1) ◽

pp. 111-124 ◽

Cited By ~ 36

Author(s):

N D Light ◽

A J Bailey

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Type I Collagen ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Type I ◽

Molecular Weight Range ◽

Carbohydrate Analysis ◽

Weight Range

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.

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Structure of cDNA clones coding for the entire preproα1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences

Biochemical Journal ◽

10.1042/bj2600509 ◽

1989 ◽

Vol 260 (2) ◽

pp. 509-516 ◽

Cited By ~ 100

Author(s):

L Ala-Kokko ◽

S Kontusaari ◽

C T Baldwin ◽

H Kuivaniemi ◽

D J Prockop

Keyword(s):

Amino Acid ◽

Type I Collagen ◽

Type Iii Collagen ◽

Cdna Clones ◽

Type I ◽

Base Pairs ◽

Type Iii ◽

Human Type ◽

Type I Procollagen ◽

Type Iii Procollagen

Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.

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Inhibition of the Activities of Matrix Metalloproteinases 2, 8, and 9 by Chlorhexidine

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.3.437-439.1999 ◽

1999 ◽

Vol 6 (3) ◽

pp. 437-439 ◽

Cited By ~ 273

Author(s):

Renée Gendron ◽

Daniel Grenier ◽

Timo Sorsa ◽

Denis Mayrand

Keyword(s):

Matrix Metalloproteinases ◽

Type I Collagen ◽

Inflammatory Diseases ◽

Human Peripheral Blood ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Inhibitory Effect ◽

Blue Staining ◽

Collagenolytic Activity ◽

Type I

ABSTRACT Matrix metalloproteinases (MMPs) are a host cell-derived proteolytic enzyme family which plays a major role in tissue-destructive inflammatory diseases such as periodontitis. The aim of the present study was to evaluate the inhibitory effect of chlorhexidine (CHX) on MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-8 (collagenase 2) activity. Heat-denatured type I collagen (gelatin) was incubated with pure human MMP-2 or -9 activated with p-aminophenylmercuric acetate (APMA), and the proteolytic degradation of gelatin was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. The effect of CHX on MMP-8 activity was also studied with a cellular model addressing the ability of phorbol myristate acetate (PMA)-triggered human peripheral blood neutrophils (polymorphonuclear leukocytes [PMNs]) to degrade native type I collagen. CHX inhibited the activities of both gelatinases (A and B), but MMP-2 appeared to be more sensitive than MMP-9. Adding calcium chloride to the assay mixtures almost completely prevented the inhibition of MMP-9 activity by CHX, while the inhibition of MMP-2 activity could be reversed only when CHX was used at a low concentration. This observation suggests that CHX may act via a cation-chelating mechanism. CHX dose-dependently inhibited collagenolytic activity of MMP-8 released by PMA-triggered PMNs. MMP-8 without APMA activation was inhibited clearly more efficiently than APMA-activated MMP-8. Our study suggests that the direct inhibition of the MMPs’ activities by CHX may represent a new valuable effect of this antimicrobial agent and explains, at least in part, the beneficial effects of CHX in the treatment of periodontitis.

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Nature of collagens synthesized by monkey periodontal-ligament fibroblasts in vitro

Biochemical Journal ◽

10.1042/bj1700063 ◽

1978 ◽

Vol 170 (1) ◽

pp. 63-71 ◽

Cited By ~ 24

Author(s):

H F Limeback ◽

J Sodek ◽

D M Brunette

Keyword(s):

Periodontal Ligament ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Type Iii Collagen ◽

Type I ◽

Periodontal Ligament Fibroblasts ◽

Type Iii ◽

Type I Procollagen

1. First subcultures of fibroblast-like cells from adult monkey periodontal ligament were incubated in the presence of 14C-labelled amino acids and produced significant amounts of type-I and type-III collagens. 2. The proportion of type-III collagen produced was calculated on the basis of the recovery of procollagens from DEAE-cellulose chromatography to be approx. 20%, and at least 10% when analysed as collagens on CM-cellulose chromatography. 3. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the procollagens, the collagens and their CNBr peptides was used to confirm the identity of the collagen types. 4. In serum-free media extensive conversion of type-I procollagen, but not of type-III procollagen, into collagen was observed, suggesting that a specific type-I procollagen peptidase was produced. 5. The pattern of collagen synthesis was not significantly different from that obtained with fibroblasts derived from skin corium of the same animals.

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