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The Journal of Biological Chemistry Aug 2022Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most...
Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly), suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.
Topics: Bacterial Proteins; Collagen; Collagenases; Hydroxyproline; Substrate Specificity; Vibrionaceae; Water; Zinc
PubMed: 35679897
DOI: 10.1016/j.jbc.2022.102109 -
Experimental Biology and Medicine... Sep 2023This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues of...
This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues of piglets with intrauterine growth restriction (IUGR), thereby contributing to a severe glycine deficiency in these compromised neonates. At 0, 7, 14, and 21 days of age, IUGR piglets were euthanized, and tissues (liver, small intestine, kidney, pancreas, stomach, skeletal muscle, and heart) were obtained for metabolic studies, as well as the determination of enzymatic activities, cell-specific localization, and expression of mRNAs for glycine-synthetic enzymes. The results indicated relatively low enzymatic activities for 4-hydroxyproline oxidase (OH-POX), proline oxidase, serine hydroxymethyltransferase, threonine dehydrogenase (TDH), alanine: glyoxylate transaminase, and 4-hydroxy-2-oxoglutarate aldolase in the kidneys and liver from 0- to 21-day-old IUGR pigs, in the pancreas of 7- to 21-day-old IUGR pigs, and in the small intestine and skeletal muscle (except TDH) of 21-day-old IUGR pigs. Accordingly, the rates of conversion of 4-hydroxyproline into glycine were relatively low in tissues of IUGR piglets. The expression of mRNAs for glycine-synthetic enzymes followed the patterns of enzymatic activities and was also low. Immunohistochemical analyses revealed the relatively low abundance of OH-POX protein in the liver, kidney, and small intestine of IUGR piglets, and the lack of OH-POX zonation in their livers. These novel results provide a metabolic basis to explain why the endogenous synthesis of glycine is insufficient for optimum growth of IUGR piglets and have important implications for improving the nutrition and health of other mammalian neonates including humans with IUGR.
Topics: Humans; Female; Animals; Swine; Fetal Growth Retardation; Animals, Newborn; Hydroxyproline; Glycine; Intestine, Small; RNA, Messenger; Mammals
PubMed: 37837389
DOI: 10.1177/15353702231199080 -
Dialysable and non-dialysable hydroxyproline in the rat's urine: age related and diurnal variations.The Journal of Physiology May 19821. Urinary dialysable and non-dialysable hydroxyproline, which are considered good indices of bone resorption and neoformation respectively, were determined in rats...
1. Urinary dialysable and non-dialysable hydroxyproline, which are considered good indices of bone resorption and neoformation respectively, were determined in rats under conditions that modify skeleton metabolism, such as body growth and parathyroid or calcitonin administration. It was also investigated whether dialysable and non-dialysable hydroxyproline excretions showed significant circadian fluctuations in rats of different ages.2. Dialysable hydroxyproline excretion sharply decreased from the first to the fifth months of age and underwent further gradual reduction up to the fourteenth month of life. Non-dialysable hydroxyproline excretion followed a smoother decrease up to the fifth month, then remained constant. Urinary excretion of non-dialysable hydroxyproline expressed as a percentage of the total hydroxyprolinuria (n.d.%) slowly increased with advancing rat age.3. In 2-, 4- and 6-month old rats, dialysable hydroxyproline excretion showed significant circadian fluctuations with minima and maxima at the end of the dark and light fraction of the cycle respectively. Daily fluctuations were greater in young and adult rats (50-65% of the respective average levels) than in 4-month old rats (25%). Non-dialysable hydroxyproline excretion followed similar but less pronounced patterns. Significant circadian fluctuations of n.d.% were detectable only in 2- and 4-month old rats, with peaks at 04.00-05.00 hr, thus indicating that the bone formation/resorption ratio increased in the nocturnal fraction of the cycle.4. Young rats administered with calcitonin exhibited reduced levels of urinary dialysable but not of non-dialysable hydroxyproline when the hormone was given at 13.30 hr. No changes were observed when calcitonin was injected at 19.30 hr. On the contrary, both diurnal and nocturnal parathyroid hormone administration to young rats caused increased levels of dialysable and non-dialysable hydroxyproline of the same magnitude.
Topics: Age Factors; Animals; Calcitonin; Circadian Rhythm; Dialysis; Hydroxyproline; Male; Parathyroid Hormone; Rats; Rats, Inbred Strains
PubMed: 7202048
DOI: 10.1113/jphysiol.1982.sp014173 -
Scientific Reports Nov 2022Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to...
Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to their discontinuous values and reader-dependent variability. Here we used an automated image analysis software to measure fibrosis deposition in two relevant preclinical models of liver fibrosis, and established correlation with other quantitative fibrosis descriptors. Longitudinal quantification of liver fibrosis was carried out during progression of post-necrotic (CCl-induced) and metabolic (HF-CDAA feeding) models of chronic liver disease in mice. Whole slide images of picrosirius red-stained liver sections were analyzed using a fully automated, unsupervised software. Fibrosis was characterized by a significant increase of collagen proportionate area (CPA) at weeks 3 (CCl) and 8 (HF-CDAA) with a progressive increase up to week 18 and 24, respectively. CPA was compared to collagen content assessed biochemically by hydroxyproline assay (HYP) and by standard histological staging systems. CPA showed a high correlation with HYP content for CCl (r = 0.8268) and HF-CDAA (r = 0.6799) models. High correlations were also found with Ishak score or its modified version (r = 0.9705) for CCl and HF-CDAA (r = 0.9062) as well as with NASH CRN for HF-CDAA (r = 0.7937). Such correlations support the use of automated digital analysis as a reliable tool to evaluate the dynamics of liver fibrosis and efficacy of antifibrotic drug candidates in preclinical models.
Topics: Mice; Animals; Liver Cirrhosis; Liver; Fibrosis; Collagen; Hydroxyproline
PubMed: 36333365
DOI: 10.1038/s41598-022-22902-w -
Biochemistry Feb 2009Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over... (Comparative Study)
Comparative Study
Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue l-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 A, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Conserved Sequence; Crystallography, X-Ray; Escherichia coli Proteins; Humans; Hydroxyproline; Membrane Proteins; Molecular Sequence Data; Proline; Proline Oxidase; Substrate Specificity; Tyrosine
PubMed: 19140736
DOI: 10.1021/bi802094k -
International Wound Journal Feb 2016The objective of this study was to evaluate the wound healing potential of L-glutamine in laboratory rats using excision and incision wound models. Excision wounds of...
The objective of this study was to evaluate the wound healing potential of L-glutamine in laboratory rats using excision and incision wound models. Excision wounds of size 500 mm(2) and depth 2 mm were made on the dorsal portion of male Wistar rats (230-250 g) and were used for the study of oral L-glutamine (1 g/kg) treatment on the rate of contraction of wound and epithelisation. Histological evaluation of wound tissue was also performed. Six-centimetre-long two linear-paravertebral incisions in male Wistar rats (230-250 g) were used to study the effect of L-glutamine (1 g/kg, p.o.) treatment on tensile strength, total protein and hydroxyproline content in the incision model. Oral administration of L-glutamine (1 g/kg) significantly decreased wound area, epithelisation period and wound index, whereas the rate of wound contraction significantly increased (P < 0·001) when compared with vehicle control rats in the excision wound model. Tensile strength, hydroxyproline content and protein level were significantly increased (P < 0·001) in L-glutamine (1 g/kg, p.o.)-treated rats when compared with vehicle control rats in the incision wound model. Histological evaluation of wound tissue from L-glutamine (1 g/kg, p.o.)-treated rats showed complete epithelialisation with new blood vessel formation and high fibrous tissues in the excision wound model. In conclusion, oral administration of l-glutamine (1 g/kg) promotes wound healing by acting on various stages of wound healing such as collagen synthesis, wound contraction and epithelialisation.
Topics: Administration, Oral; Animals; Glutamine; Hydroxyproline; Male; Neovascularization, Physiologic; Proteins; Rats, Wistar; Re-Epithelialization; Skin; Tensile Strength; Wound Healing; Wounds and Injuries
PubMed: 24690128
DOI: 10.1111/iwj.12246 -
Ultrasonics Sonochemistry Sep 2022The effect of ultrasound treatment (300 W; 0, 5, 10, 15, 20 and 25 min) on the extractability of acid soluble collagen from yellowfin tuna skin and its structural,...
The effect of ultrasound treatment (300 W; 0, 5, 10, 15, 20 and 25 min) on the extractability of acid soluble collagen from yellowfin tuna skin and its structural, physicochemical and functional properties were investigated. Ultrasound treatments significantly increased collagen extraction yield from the tuna skin up to 2.7 times, compared to the conventional extraction with acetic acid. The level of proline, hydroxyproline and thermal stability of collagens increased by applying ultrasound while their native triple-stranded helical structure was well-preserved, as revealed by X-ray diffraction and FTIR spectroscopy. However, ultrasound treatment reduced the particle size of the collagens which increased their pH and salt induced solubility. The water holding capacity and the emulsifying properties of ultrasound treated collagens were also higher than those produced with the conventional method. Altogether, the results suggested that ultrasonication can be a promising assistant technology for improving native collagen extraction efficiency from tuna skin and its functionality but its duration should be carefully optimized.
Topics: Animals; Collagen; Hydroxyproline; Solubility; Tuna; Water
PubMed: 36007329
DOI: 10.1016/j.ultsonch.2022.106129 -
Journal of Tissue Engineering and... Feb 2017Scaffold-free engineered cartilage is being explored as a treatment for osteoarthritis. In this study, frictional shear stress was applied to determine the friction and...
Scaffold-free engineered cartilage is being explored as a treatment for osteoarthritis. In this study, frictional shear stress was applied to determine the friction and damage behaviour of scaffold-free engineered cartilage, and tissue composition was investigated as it related to damage. Scaffold-free engineered cartilage frictional shear stress was found to exhibit a time-varying response similar to that of native cartilage. However, damage occurred that was not seen in native cartilage, manifesting primarily as tearing through the central plane of the constructs. In engineered cartilage, cells occupied a significantly larger portion of the tissue in the central region where damage was most prominent (18 ± 3% of tissue was comprised of cells in the central region vs 5 ± 1% in the peripheral region; p < 0.0001). In native cartilage, cells comprised 1-4% of tissue for all regions. Average bulk cellularity of engineered cartilage was also greater (68 × 10 ± 4 × 10 vs 52 × 10 ± 22 × 10 cells/mg), although this difference was not significant. Bulk tissue comparisons showed significant differences between engineered and native cartilage in hydroxyproline content (8 ± 2 vs 45 ± 3 µg HYP/mg dry weight), solid content (12.5 ± 0.4% vs 17.9 ± 1.2%), shear modulus (0.06 ± 0.02 vs 0.15 ± 0.07 MPa) and aggregate modulus (0.12 ± 0.03 vs 0.32 ± 0.14 MPa), respectively. These data indicate that enhanced collagen content and more uniform extracellular matrix distribution are necessary to reduce damage susceptibility. Copyright © 2014 John Wiley & Sons, Ltd.
Topics: Animals; Bioreactors; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagen; Extracellular Matrix; Friction; Hydroxyproline; Osteoarthritis; Pressure; Rabbits; Stress, Mechanical; Surface Properties; Tissue Engineering
PubMed: 24965503
DOI: 10.1002/term.1925 -
Marine Drugs Nov 2023The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine;...
Low-Molecular-Weight Fish Collagen Peptide (Valine-Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine-Glycine) Prevents Osteoarthritis Symptoms in Chondrocytes and Monoiodoacetate-Injected Rats.
The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine; LMWCP) on HO- or LPS-treated primary chondrocytes and monoiodoacetate (MIA)-induced osteoarthritis rat models. Our findings indicated that LMWCP treatment exhibited protective effects by preventing chondrocyte death and reducing matrix degradation in both HO-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. This was achieved by increasing the levels of aggrecan, collagen type I, collagen type II, TIMP-1, and TIMP-3, while simultaneously decreasing catabolic factors such as phosphorylation of Smad, MMP-3, and MMP-13. Additionally, LMWCP treatment effectively suppressed the activation of inflammation and apoptosis pathways in both LPS-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. These results suggest that LMWCP supplementation ameliorates the progression of osteoarthritis through its direct impact on inflammation and apoptosis in chondrocytes.
Topics: Rats; Animals; Chondrocytes; Hydroxyproline; Glycine; Hydrogen Peroxide; Lipopolysaccharides; Osteoarthritis; Inflammation; Collagen Type II; Peptides; Valine; Cartilage, Articular; Cells, Cultured
PubMed: 38132929
DOI: 10.3390/md21120608 -
Kidney International Mar 2002Over 54 generations, we have successfully bred a strain of rats that maximizes urinary calcium excretion. The rats now consistently excrete 8 to 10 times as much calcium...
BACKGROUND
Over 54 generations, we have successfully bred a strain of rats that maximizes urinary calcium excretion. The rats now consistently excrete 8 to 10 times as much calcium as controls, uniformly form poorly crystalline calcium phosphate kidney stones, and are termed genetic hypercalciuric stone-forming (GHS) rats. These rats were used to test the hypothesis that increasing urinary oxalate excretion would not only increase the supersaturation with respect to the calcium oxalate solid phase, but also would increase the ratio of calcium oxalate-to-calcium phosphate supersaturation and result in calcium oxalate stone formation.
METHODS
To increase urine oxalate excretion an oxalate precursor, hydroxyproline, was added to the diet of male GHS rats. The GHS rats were fed a standard 1.2% calcium diet alone or with 1%, 3% or 5% trans-4-hydroxy-l-proline (hydroxyproline).
RESULTS
The addition of 1% hydroxyproline to the diet of GHS rats led to an increase in urinary oxalate excretion, which did not increase further with the provision of additional hydroxyproline. The addition of 1% and 3% hydroxyproline did not alter calcium excretion while the provision of 5% hydroxyproline led to a decrease in urine calcium excretion. The addition of 1% hydroxyproline led to an increase in urinary calcium oxalate supersaturation, which did not further increase with additional hydroxyproline. The addition of 1% and 3% hydroxyproline did not alter urinary supersaturation with respect to calcium hydrogen phosphate while the addition of 5% hydroxyproline tended to lower this supersaturation. Compared to rats fed the control and the 3% hydroxyproline diet the addition of 5% hydroxyproline increased the ratio of calcium oxalate supersaturation to calcium phosphate supersaturation. Virtually all rats formed stones. In the control and 1% hydroxyproline group, all of the stones were composed of calcium and phosphate (apatite), in the 3% hydroxyproline group the stones were a mixture of apatite and calcium oxalate, while in the 5% hydroxyproline group all of the stones were calcium oxalate.
CONCLUSIONS
The provision of additional dietary hydroxyproline to GHS rats increases urinary oxalate excretion, calcium oxalate supersaturation and the ratio of calcium oxalate-to-calcium phosphate supersaturation, resulting in the formation of calcium oxalate kidney stones. Thus, with the addition of a common amino acid, the GHS rats now not only model the most common metabolic abnormality found in patients with nephrolithiasis, hypercalciuria, but form the most common type of kidney stone, calcium oxalate.
Topics: Animals; Calcium; Calcium Oxalate; Calcium Phosphates; Diet; Diuresis; Hydrogen-Ion Concentration; Hydroxyproline; Ions; Kidney; Kidney Calculi; Male; Microscopy, Electron, Scanning; Rats; Rats, Sprague-Dawley; Urine
PubMed: 11849452
DOI: 10.1046/j.1523-1755.2002.00190.x