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The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of l-lysine by l-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational l-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinked.

Glutaric acidemia type I (GA-I) is an inborn error of metabolism of lysine, hydroxylysine, and tryptophan, caused by glutaryl-CoA-dehydrogenase (GCDH) deficiency, characterized by the buildup of toxic organic acids predominantly in the brain. After acute catabolic states, patients usually develop striatal degeneration, but the mechanisms behind this damage are still unknown. Quinolinic acid (QA), a metabolite of the kynurenine pathway, increases especially during infections/inflammatory processes, and could act synergically with organic acids, contributing to the neurological features of GA-I. The aim of this study was to investigate whether QA increases seizure susceptibility and modifies brain oscillation patterns in an animal model of GA-I, the Gcdh-/- mice taking high-lysine diet (Gcdh-/- -Lys). Therefore, the characteristics of QA-induced seizures and changes in brain oscillatory patterns were evaluated by video-electroencephalography (EEG) analysis recorded in Gcdh-/- -Lys, Gcdh+/+ -Lys, and Gcdh-/- -N (normal diet) animals. We found that the number of seizures per animal was similar for all groups receiving QA, Gcdh-/- -Lys-QA, Gcdh+/+ -Lys-QA, and Gcdh-/- -N-QA. However, severe seizures were observed in the majority of Gcdh-/- -Lys-QA mice (82%), and only in 25% of Gcdh+/+ -Lys-QA and 44% of Gcdh-/- -N-QA mice. All Gcdh-/- -Lys animals developed spontaneous recurrent seizures (SRS), but Gcdh-/- -Lys-QA animals had increased number of SRS, higher mortality rate, and significant predominance of lower frequency oscillations on EEG. Our results suggest that QA plays an important role in the neurological features of GA-I, as Gcdh-/- -Lys mice exhibit increased susceptibility to intrastriatal QA-induced seizures and long-term changes in brain oscillations.

In spite of major advances over the past several decades in diagnosis and treatment, breast cancer remains a global cause of morbidity and premature death for both human and veterinary patients. Due to multiple shared clinicopathological features, dogs provide an excellent model of human breast cancer, thus, a comparative oncology approach may advance our understanding of breast cancer biology and improve patient outcomes. Despite an increasing awareness of the critical role of fibrillar collagens in breast cancer biology, tumor-permissive collagen features are still ill-defined. Here, we characterize the molecular and morphological phenotypes of type I collagen in canine mammary gland tumors. Canine mammary carcinoma samples contained longer collagen fibers as well as a greater population of wider fibers compared to non-neoplastic and adenoma samples. Furthermore, the total number of collagen cross-links enriched in the stable hydroxylysine-aldehyde derived cross-links was significantly increased in neoplastic mammary gland samples compared to non-neoplastic mammary gland tissue. The mass spectrometric analyses of type I collagen revealed that in malignant mammary tumor samples, lysine residues, in particular those in the telopeptides, were markedly over-hydroxylated in comparison to non-neoplastic mammary tissue. The extent of glycosylation of hydroxylysine residues was comparable among the groups. Consistent with these data, expression levels of genes encoding lysyl hydroxylase 2 (LH2) and its molecular chaperone FK506-binding protein 65 were both significantly increased in neoplastic samples. These alterations likely lead to an increase in the LH2-mediated stable collagen cross-links in mammary carcinoma that may promote tumor cell metastasis in these patients.

The aim of the research is a qualitative and quantitative study of the amino acid composition of Syrian rue (Peganum harmala, Nitrariaceae), growing in the natural conditions of the Azerbaijan Republic. After preliminary confirmation of the presence of amino acids, the plant parts were subjected to in-depth study by ion-exchange chromatography using post-column derivatization on a L-8800 amino acid analyzer (Hitachi, Ltd.). Amino acid analysis of the roots of P. harmala, conducted in the course of this study, was carried out by us for the first time. According to the results of the analysis, 18 amino acids were identified in the studied parts of P.harmala, 8 of which nonessential, 9 are essential and 3 are conditionally essential. The total amount of amino acids for the roots was 7.162%, seeds - 6.096%, and stems - 14.676%. From the individual amino acids in the underground organs of P. harmala, proline predominates (2.149%), and aspartic acid predominates in the stems and seeds (2.698% and 2.394%, respectively). The least detected amino acids are ornithine in the roots and stems (0.007% and 0.020%, respectively) and cysteine in the seeds (0.024%). Hydroxyproline was not found in the stems. At the same time, with the exception of proline, hydroxyproline, hydroxylysine and ornithine in the stems, the remaining amino acids were found to be higher than in other organs.

Pullulan, a naturally occurring polysaccharide was oxidized using sodium periodate and converted into its aldehyde derivative and used as a bio crosslinker for stabilizing collagen. The crosslinking mainly occurred due to Schiff's base reaction between amino group of lysine or hydroxylysine present in collagen with the aldehyde groups of oxidized pullulan. A known concentration of collagen was crosslinked with different concentrations of oxidized pullulan and scaffolds were prepared by freeze drying method. The crosslinking efficiency and biodegradation of the crosslinked scaffolds showed higher degree of crosslinking and slower degradation rate with increase in cross linker concentration. The surface morphology of scaffolds using SEM showed that the scaffolds with 3% concentration of crosslinker (Col-OxP-3) exhibited highly porous nature with interconnecting pores of 66.35 μm size compared to other groups, which was also supported by porosity and tensile strength measurement. The tensile strength was 10 times higher in Col-OxP-3 scaffold compared to native collagen. The swelling capacity of the scaffolds increased with increase in crosslinker concentration and the equilibrium was retained up to 60 min. The crosslinked scaffolds were non-toxic to 3T3 fibroblast cell line proving their biocompatible nature. Thus, this new class of bio-crosslinker can be used in replacement of synthetic crosslinkers for biomaterial synthesis.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Introduction: Hydroxylation is one of the most important post-translational modifications (PTM) in cellular functions and is linked to various diseases. The addition of one of the hydroxyl groups (OH) to the lysine sites produces hydroxylysine when undergoes chemical modification. Methods: The method which is used in this study for identifying hydroxylysine sites based on powerful mathematical and statistical methodology incorporating the sequence-order effect and composition of each object within protein sequences. This predictor is called "iHyd-LysSite (EPSV)" (identifying hydroxylysine sites by extracting enhanced position and sequence variant technique). The prediction of hydroxylysine sites by experimental methods is difficult, laborious and highly expensive. In silico technique is an alternative approach to identify hydroxylysine sites in proteins. Results: The experimental results require that the predictive model should have high sensitivity and specificity values and must be more accurate. The self-consistency, independent, 10-fold crossvalidation and jackknife tests are performed for validation purposes. These tests are resulted by using three renowned classifiers, Neural Networks (NN), Random Forest (RF) and Support Vector Machine (SVM) with the demanding prediction rate. The overall predictive outcomes are extraordinarily superior to the results obtained by previous predictors. The proposed model contributed an excellent prediction rate in the system for NN, RF, and SVM classifiers. The sensitivity and specificity results using all these classifiers for jackknife test are 96.08%, 94.99%, 98.16% and 97.52%, 98.52%, 80.95%. Conclusion: The results obtained by the proposed tool show that this method may meet the future demand of hydroxylysine sites with a better prediction rate over the existing methods.

Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism. A defect of glutaryl-CoA dehydrogenase results in the accumulation of 3-hydroxyglutaric acid and glutaric acid. Nutrition management of GA-1 consists of restricting lysine and tryptophan, supplementing L-carnitine, and providing sufficient energy to prevent catabolism. Patients with GA-1 have a particularly high risk of permanent cerebral damage from a metabolic crisis.

Highlights • Tendon and ligament collagens differ in their post-translational lysine and cross-linking chemistry. • In ligament collagen, hydroxylysyl aldehyde, permanent cross-linking dominates. • Tendon collagen has a mix of cross-links based on lysyl and hydroxylysyl aldehydes. • The profile in tendon appears more adapted to facilitating growth, structural remodeling and repair of the fibrillar matrix., Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.

Graphical abstract, Highlights • Ultrasound treatment promoted myofibril fragmentation of chicken gizzard. • Ultrasound treatment could weaken the degree of covalent cross-linking of collagen. • Ultrasound treatment reduced the thermal stability of chicken gizzard collagen. • 500 W/30 min ultrasound treatment had the best tenderization effect., The tenderizing effect of different ultrasound treatments on the characteristics of muscle fibers and connective tissue of chicken gizzard was investigated. It could be concluded that the shear force and muscle fiber diameter of the sample treated with ultrasound for 500 W/30 min were decreased by 27.1% and 26.2%, respectively, while the myofibril fragmentation index (MFI) was increased by 238.1% than the control. More importantly, the contents of hydroxylysine pyridinoline and lysine pyridinoline of the samples treated with ultrasound for 500 W/30 min were 23.1% and 40.5% lower than those of the control. Tenderizing effect of 500 W/30 min sample on thermal stability was verified from the decrease in transition temperature (Tmax) (10.7%) and enthalpy (ΔH) (21.7%) of collage compared with the control. In general, proper ultrasound treatment could effectively improve the tenderness of gizzard, and 500 W/30 min had the best tenderization effect. Therefore, the treatment of ultrasound was considered as a promising and efficient technique in meat processing, especially for the meat tenderization.

Heritable thoracic aortic disease and familial thoracic aortic aneurysm/dissection are important causes of human morbidity/mortality, most without identifiable genetic cause. In a family with familial thoracic aortic aneurysm/dissection, we identified a missense p. (Ser178Arg) variant in PLOD1 segregating with disease, and evaluated PLOD1 enzymatic activity, collagen characteristics and in human aortic vascular smooth muscle cells, studied the effect on function. Comparison with homologous PLOD3 enzyme indicated that the pathogenic variant may affect the N-terminal glycosyltransferase domain, suggesting unprecedented PLOD1 activity. In vitro assays demonstrated that wild-type PLOD1 is capable of processing UDP-glycan donor substrates, and that the variant affects the folding stability of the glycosyltransferase domain and associated enzymatic functions. The PLOD1 substrate lysine was elevated in the proband, however the enzymatic product hydroxylysine and total collagen content was not different, albeit despite collagen fibril narrowing and preservation of collagen turnover. In VSMCs overexpressing wild-type PLOD1, there was upregulation in procollagen gene expression (secretory function) which was attenuated in the variant, consistent with loss-of-function. In comparison, si-PLOD1 cells demonstrated hypercontractility and upregulation of contractile markers, providing evidence for phenotypic switching. Together, the findings suggest that the PLOD1 product is preserved, however newly identified glucosyltransferase activity of PLOD1 appears to be affected by folding stability of the variant, and is associated with compensatory vascular smooth muscle cells phenotypic switching to support collagen production, albeit with less robust fibril girth. Future studies should focus on the impact of PLOD1 folding/variant stability on the tertiary structure of collagen and ECM interactions.

Recent studies demonstrate the involvement of inflammatory processes in the development of depression and the anti-inflammatory effects of antidepressants. Infiltration and adhesion of neutrophils to nerve tissues and their aggressive secretion are considered as possible causes of inflammatory processes in depression. We studied the effect of the antidepressant imipramine on the adhesion and accompanied secretion of neutrophils under control conditions and in the presence of lipopolysaccharides (LPS). As a model of integrin-dependent neutrophil infiltration into tissues, we used integrin-dependent adhesion of neutrophils to the fibronectin-coated substrate. We studied the effect of the antidepressant imipramine on the adhesion of neutrophils to fibronectin and the accompanying production of reactive oxygen species (ROS) and the secretion of proteins and free amino acids under control conditions and in the presence of lipopolysaccharides (LPS). Our data showed that imipramine Imipramine inhibiteds neutrophil adhesion and concomitant secretion of proteins, including matrix metalloproteinase 9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL), which modify the extracellular matrix and basement membranes required for cell migration. Imipramine also significantly and selectively blocked the release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, an enzyme that affects the organization of the extracellular matrix by modifying collagen lysine residues. In contrast, imipramine enhanced the release of ROS by neutrophils during adhesion to fibronectin and stimulated apoptosis. The anti-inflammatory effect of imipramine may be associated with the suppression of neutrophil infiltration and their adhesion to nerve tissues by inhibiting the secretion of neutrophils, which provides these processes.

Fibrosis is a pathological state characterized by excessive deposition of the extracellular matrix components leading to impaired tissue function in the affected organ. It results in scarring of the affected tissue akin to an over-healing wound as a consequence of chronic inflammation and repair in response to injury. Persistent trauma of susceptible oral mucosa due to habitual chewing of betel quid resulting in zealous healing of the mucosal tissue is one plausible explanation for the onset of oral submucous fibrosis (OSF). The irreversibility and resistance of collagen to degradation and its high potential to undergo malignant change are a major reason for morbidity in OSF. Hence, early diagnosis and timely treatment are crucial to prevent the progression of OSF to malignancy. This review focuses on the mechanistic insight into the role of collagen cross-links in advancing fibrosis and possible therapeutic targets that bring about a reversal of fibrosis. These options may be beneficial if attempted as a specific therapeutic modality in OSF as is in organ fibrosis. The upregulation of lysyl oxidase and lysyl hydroxylase has been shown to exhibit the higher levels of the hydroxylysine aldehyde-derived cross-links in fibrosis and tumor stroma promoting the tumor cell survival, resistance, and invasion. The in silico analysis highlights the potential drugs that may target the genes regulating collagen crosslinking.

The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.

Intake of dietary sources of collagen may support the synthesis of collagen in varying tissues, with the availability of key amino acids being a likely contributor to its effectiveness. This study analyzed commonly consumed preparations of bone broth (BB) to assess the amount and consistency of its amino acid content. Commercial and laboratory-prepared samples, made with standardized and variable (nonstandardized) protocols, were analyzed for key amino acids (glycine, lysine, proline, leucine, hydroxyproline, and hydroxylysine). The main finding of this study was that amino acid concentrations in BB made to a standardized recipe were significantly lower for hydroxyproline, glycine, and proline (p = .003) and hydroxylysine, leucine, and lysine (p = .004) than those provided by a potentially therapeutic dose (20 g) of reference collagen supplements (p > .05). There was a large variability in the amino acid content of BB made to nonstandardized recipes, with the highest levels of all amino acids found within the café-prepared varieties. For standardized preparations, commercial BBs were lower in all amino acids than the self-prepared varieties. There were no differences (p > .05) in the amino acid content of different batches of BB when prepared according to a standardized recipe. If the intake of collagen precursors is proven to support the synthesis of new collagen in vivo, it is unlikely that BB can provide a consistently reliable source of key amino acids. Research on the provision of key amino acids from dietary sources should continue to focus on the standard sources currently being researched.

Neutrophils release branched-chain (valine, isoleucine, leucine), aromatic (tyrosine, phenylalanine) and positively charged free amino acids (arginine, ornithine, lysine, hydroxylysine, histidine) when adhere and spread onto fibronectin. In the presence of agents that impair cell spreading or adhesion (cytochalasin D, fMLP, nonadhesive substrate), neutrophils release the same amino acids, except for a sharp decrease in hydroxylysine and an increase in phenylalanine, indicating their special connection with cell adhesion. Plasma of patients with diabetes is characterized by an increased content of branched-chain and aromatic amino acids and a reduced ratio of arginine/ornithine compared to healthy human plasma. Our data showed that the secretion of neutrophils, regardless of their adhesion state, can contribute to this shift in the amino acid content. Abbreviations: BCAAs: branched-chain amino acids; Е2: 17β-estradiol; LPS: lipopolysaccharide from Salmonella enterica serovar Typhimurium; fMLP: N-formylmethionyl-leucyl-phenylalanine.

During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.

Background The arteriovenous fistula (AVF) is the preferred hemodialysis access for end-stage renal disease (ESRD) patients. Yet, establishment of a functional AVF presents a challenge, even for the most experienced surgeons, since postoperative stenosis frequently occludes the AVF. Stenosis results from the loss of compliance in fibrotic areas of the fistula which turns intimal hyperplasia into an occlusive feature. Fibrotic remodeling depends on deposition and crosslinking of collagen by lysyl oxidase (LOX), an enzyme that catalyzes the deamination of lysine and hydroxylysine residues, facilitating intra/intermolecular covalent bonds. We postulate that pharmacological inhibition of lysyl oxidase (LOX) increases postoperative venous compliance and prevents stenosis in a rat AVF model. Methods LOX gene expression and vascular localization were assayed in rat AVFs and human pre-access veins, respectively. Collagen crosslinking was measured in humans AVFs that matured or failed, and in rat AVFs treated with β-aminopropionitrile (BAPN), an irreversible LOX inhibitor. BAPN was either injected systemically or delivered locally around rat AVFs using nanofiber scaffolds. The major endpoints were AVF blood flow, wall fibrosis, collagen crosslinking, and vascular distensibility. Results Non-maturation of human AVFs was associated with higher LOX deposition in pre-access veins (N=20, P=0.029), and increased trivalent crosslinks (N=18, P=0.027) in human AVF tissues. Systemic and local inhibition of LOX increased AVF distensibility, while reducing wall fibrosis and collagen crosslinking in rat fistulas. Conclusions Our results demonstrate that BAPN-mediated inhibition of LOX significantly improves vascular remodeling in experimental fistulas.

Collagen is a major constituent of the extracellular matrix (ECM) that confers fundamental mechanical properties to tissues. To allow proper folding in triple-helices and organization in quaternary super-structures, collagen molecules require essential post-translational modifications (PTMs), including hydroxylation of proline and lysine residues, and subsequent attachment of glycan moieties (galactose and glucose) to specific hydroxylysine residues on procollagen alpha chains. The resulting galactosyl-hydroxylysine (Gal-Hyl) and less abundant glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) are amongst the simplest glycosylation patterns found in nature and are essential for collagen and ECM homeostasis. These collagen PTMs depend on the activity of specialized glycosyltransferase enzymes. Although their biochemical reactions have been widely studied, several key biological questions about the possible functions of these essential PTMs are still missing. In addition, the lack of three-dimensional structures of collagen glycosyltransferase enzymes hinders our understanding of the catalytic mechanisms producing this modification, as well as the impact of genetic mutations causing severe connective tissue pathologies. In this mini-review, we summarize the current knowledge on the biochemical features of the enzymes involved in the production of collagen glycosylations and the current state-of-the-art methods for the identification and characterization of this important PTM.

The fracture resistance of cortical bone and matrix hydration are known to decline with advanced aging. However, the underlying mechanisms remain poorly understood, and so we investigated levels of matrix proteins and post-translational modifications (PTM) of collagen I in extracts from the tibia of 6-mo. and 20-mo. old BALB/c mice (female and male analysis done separately). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the levels of collagen I deamidation at specific asparagine (Asn) and glutamine (Gln) residues significantly increased with age. Other non-enzymatic PTMs such as carboxymethylation of lysine (CML) were detected as well, but the relative abundance did not vary with age. No significant age-related differences in the abundance of hydroxylysine glycosylation sites were found, but hydroxylation levels at a few of the numerous lysine and proline hydroxylation sites significantly changed by a small amount with age. We performed molecular modeling and dynamics (MD) simulations for three triple helical fragments representing collagen I regions with prominent age-dependent increases in deamidation as identified by LC-MS/MS of male extracts. These 3 fragments included deamidated Asn and Gln residues as follows: 1) an Asn(428) site of the α2(I) chain in which deamidation levels increased from 4.4% at 6-mo. to 8.1% at 20-mo., 2) an Asn(983) site of the α2(I) chain with a deamidation increase from 18.3% to 36.8% with age and an Asn(1052) site of the α1(I) chain with consistent deamidation levels of ~60% across the age groups, and 3) a Gln(410) site of the α1(I) chain that went from no detectable deamidation at 6-mo. to 2.7% at 20-mo. and a neighboring Asn(421) site of the same chain with an age-related deamidation increase from 3.6% to 16.3%. The deamidation levels at these sites inversely correlated with an estimate of toughness determined from three-point bending tests of the femur mid-diaphysis. MD revealed that the sidechains become more negatively charged at deamidated sites and that deamidation alters hydrogen bonding with water along the collagen backbone while increasing water interactions with the aspartic and glutamic acid sidechains. Our findings suggest a new mechanism of the age-dependent reduction in the fracture resistance of cortical bone whereby deamidation of Asn and Glu residues redistributes bound water within collagen I triple helix.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine ((79)Br, (81)Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Collagen deposition and stromal stiffening accompany malignancy, compromise treatment, and promote tumor aggression. Clarifying the molecular nature of and the factors that regulate extracellular matrix stiffening in tumors should identify biomarkers to stratify patients for therapy and therapeutic interventions to improve outcome. We profiled lysyl hydroxylase- and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in more aggressive human breast cancer subtypes with the stiffest stroma. These tissues also harbored the highest number of tumor-associated macrophages (TAM), whose therapeutic ablation not only reduced metastasis, but also concomitantly decreased accumulation of collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme lysyl oxidase had no impact on collagen crosslinking in PyMT mammary tumors, whereas stromal cell targeting did. Consistently, stromal cells in microdissected human tumors expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of a cohort of breast cancer patient biopsies revealed that stromal expression of lysyl hydroxylase two, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening correlated significantly disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumor aggression and identify lysyl hydroxylase two as a novel stromal biomarker.SignificanceWe show infiltrating macrophages induce stromal fibroblast, and not epithelial, expression of collagen crosslinking enzymes that drive tumor stiffening. Stromal enzyme LH2 is significantly upregulated in breast cancer patients with the stiffest stroma, the most trivalent HLCCs and the worst prognosis, underscoring its potential as a biomarker and therapeutic target.

Collagen undergoes many types of post-translational modifications (PTMs), including intracellular modifications and extracellular modifications. Among these PTMs, glycosylation of hydroxylysine (Hyl) is the most complicated. Experimental studies demonstrated that this PTM ceases once the collagen triple helix is formed and that Hyl-O-glycosylation modulates collagen fibrillogenesis. However, the underlying atomic-level mechanisms of these phenomena remain unclear. In this study, we first adapted the force field parameters for O-linkages between Hyl and carbohydrates and then investigated the influence of Hyl-O-glycosylation on the structure of type I collagen molecule, by performing comprehensive molecular dynamic simulations in explicit solvent of collagen molecule segment with and without the glycosylation of Hyl. Data analysis demonstrated that (i) collagen triple helices remain in a triple-helical structure upon glycosylation of Hyl; (ii) glycosylation of Hyl modulates the peptide backbone conformation and their solvation environment in the vicinity and (iii) the attached sugars are arranged such that their hydrophilic faces are well exposed to the solvent, while their hydrophobic faces point towards the hydrophobic portions of collagen. The adapted force field parameters for O-linkages between Hyl and carbohydrates will aid future computational studies on proteins with Hyl-O-glycosylation. In addition, this work, for the first time, presents the detailed effect of Hyl-O-glycosylation on the structure of human type I collagen at the atomic level, which may provide insights into the design and manufacture of collagenous biomaterials and the development of biomedical therapies for collagen-related diseases.

Lysyl hydroxylase 2 (LH2) is an enzyme that catalyzes the hydroxylation of lysine (Lys) residues in fibrillar collagen telopeptides, a critical post-translational modification for the stability of intermolecular cross-links. Though abnormal LH2 activities have been implicated in various diseases including Bruck syndrome, the molecular basis of the pathologies is still not well understood. Since LH2 null mice die at early embryonic stage, we generated LH2 heterozygous (LH2+/-) mice in which LH2 level is significantly diminished, and characterized collagen and bone phenotypes using femurs. Compared to the wild-type (WT), LH2+/- collagen showed a significant decrease in the ratio of hydroxylysine (Hyl)- to the Lys-aldehyde-derived collagen cross-links without affecting the total number of aldehydes involved in cross-links. Mass spectrometric analysis revealed that, in LH2+/- type I collagen, the extent of hydroxylation of all telopeptidyl Lys residues was significantly decreased. In the helical domain, Lys hydroxylation at the cross-linking sites was either unaffected or slightly lower, but other sites were significantly diminished compared to WT. In LH2+/- femurs, mineral densities of cortical and cancellous bones were significantly decreased and the mechanical properties of cortical bones evaluated by nanoindentation analysis were compromised. When cultured, LH2+/- osteoblasts poorly produced mineralized nodules compared to WT osteoblasts. These data provide insight into the functionality of LH2 in collagen molecular phenotype and its critical role in bone matrix mineralization and mechanical properties.

Collagen is the most abundant protein in humans and is heavily post-translationally modified. Its biosynthesis is very complex and requires three different types of hydroxylation (two for proline and one for lysine) that are generated in the rough endoplasmic reticulum (rER). These processes involve many enzymes and chaperones which were collectively termed the molecular ensemble for collagen biosynthesis. However, the function of some of the proteins in this molecular ensemble is controversial. While prolyl 3-hydroxylase 1 and 2 (P3H1, P3H2) are bona fide collagen prolyl 3-hydroxylases, the function of prolyl 3-hydroxylase 3 (P3H3) is less clear. A recent study of P3H3 null mice demonstrated that this enzyme had no activity as prolyl 3-hydroxylase but may instead act as a chaperone for lysyl hydroxylase 1 (LH1). LH1 is required to generate hydroxylysine for crosslinking within collagen triple helical sequences. If P3H3 is a LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should have similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared lysyl hydroxylation in type I and V collagen from P3H3 and LH1 null mice. Our results indicate LH1 plays a global role for lysyl hydroxylation in triple helical domain of type I collagen while P3H3 is indeed involved in lysyl hydroxylation particularly at crosslink formation sites but is not required for all lysyl hydroxylation sites in type I collagen triple helix. Furthermore, although type V collagen from LH1 null mice surprisingly contained as much hydroxylysine as type V collagen from wild type, the amount of hydroxylysine in type V collagen was clearly suppressed in P3H3 null mice. In summary, our study suggests that P3H3 and LH1 likely have two distinct mechanisms to distinguish crosslink formation sites from other sites in type I collagen and to recognize different collagen types in the rER.Author summaryCollagen is one of the most heavily post-translationally modified proteins in the human body and its post-translational modifications provide biological functions to collagen molecules. In collagen post-translational modifications, crosslink formation on a collagen triple helix adds important biomechanical properties to the collagen fibrils and is mediated by hydroxylation of very specific lysine residues. LH1 and P3H3 show the similar role in lysine hydroxylation for specific residues at crosslink formation sites of type I collagen. Conversely, they have very distinct rules in lysine hydroxylation at other residues in type I collagen triple helix. Furthermore, they demonstrate preferential recognition and modification of different collagen types. Our findings provide a better understanding of the individual functions of LH1 and P3H3 in the rER and also offer new directions for the mechanism of lysyl hydroxylation followed by crosslink formation in different tissues and collagens.

Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M−1 s−1 (urate) to (4.8 ± 0.1) × 105 M−1 s−1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M−1 s−1 (tyrosine) to (7.7 ± 0.1) × 103 M−1 s−1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

Hydroxylysine glycosylations are collagen-specific post-translational modifications essential for maturation and homeostasis of fibrillar as well as non-fibrillar collagen molecules. Lysyl hydroxylase 3 (LH3) is the only human enzyme capable of performing two chemically-distinct collagen glycosyltransferase reactions using the same catalytic site: inverting beta-1,O-galactosylation of hydroxylysines and retaining alpha-1,2-glycosylation of galactosyl hydroxylysines. Here, we used structure-based mutagenesis to show that both glycosyltransferase activities are strongly dependent on a broad cooperative network of amino acid side chains which includes the first-shell environment of the glycosyltransferase catalytic site and shares features with both retaining and inverting enzymes. We identified critical “hot spots” leading to selective loss of inverting activity without affecting the retaining reaction. Finally, we present molecular structures of LH3 in complex with UDP-sugar analogs which provide the first structural templates for LH3 glycosyltransferase inhibitor development. Collectively, our data provide a comprehensive overview of the complex network of shapes, charges and interactions that enable LH3 glycosyltransferase activities, expanding the molecular framework for the manipulation of glycosyltransferase functions in biomedical applications. SIGNIFICANCE STATEMENT Multifunctional collagen lysyl hydroxylase-glycosyltransferase LH3 is essential for biosynthesis and maturation of collagen molecules and has strong implications in rare developmental diseases as well as in metastatic spreading of solid tumors. LH3 is the only human enzyme capable of carrying out two different glycosyltransferase reaction mechanisms within the same catalytic site to produce glucosyl-galactosyl-hydroxylysine, the most ubiquitous glycosylation pattern found in nature. This work elucidates the molecular networks enabling the LH3 glycosyltransferase reactions, highlighting a highly cooperative network of amino acid residues that concertedly ensure collagen glycosylation, and suggesting that the LH3 molecular organization may be the progenitor of more specialized glycosyltransferases. In addition, this work presents the first two small molecule scaffolds capable of modulating LH3 glycosyltransferase activities.

Eighty-two complete and partial human skeletons were excavated from a burial site on the island of Borkum in the North Sea. The site covers a period of 400years from the middle of the 14th century AD until 1720AD. The position of the skeletons in situ suggests rapid disposal of the bodies. About 75% of the skeletons were male and of a quite robust constitution, and about 60% of all individuals died rather young between 20 and 40years of age. All male skeletons exhibited particular pathological alterations as a result of the typical daily activities of seafaring men, including severe degenerative joint disease and pronounced osteochondropathies. In juvenile skeletons, these pathologies were accompanied by inflammatory processes without exception. Palaeodietary reconstruction by stable isotope analysis resulted in a protein rich diet, confirming fishing as an important part of the subsistence economy. The juveniles derived substantially more protein from plant food than the adults. Bone collagen amino acid profiles were short of hydroxylysine, suggesting that the conspicuous pathological lesions could also have been caused by a systemic connective tissue disease. Possible etiology of the disease is discussed and related to both the uneven sex ratio of the inhumations and the burial context.

Collagen in the body is exposed to a range of influences, including free radicals, which can lead to a significant change in its structure. Modeling such an effect on collagen fibrils will allow one to get a native structure in vitro, which is important for modern tissue engineering. The aim of this work is to study the effect of free radicals on a solution of hydrogen peroxide with a concentration of 0.006–0.15% on the structure of collagen fibrils in vitro, and the response of cells to such treatment. SEM measurements show a decrease in the diameter of the collagen fibrils with an increase in the concentration of hydrogen peroxide. Such treatment also leads to an increase in the wetting angle of the collagen surface. Fourier transform infrared spectroscopy demonstrates a decrease in the signal with wave number 1084 cm−1 due to the detachment of glucose and galactose linked to hydroxylysine, connected to the collagen molecule through the -C-O-C- group. During the first day of cultivating ASCs, MG-63, and A-431 cells, an increase in cell adhesion on collagen fibrils treated with H2O2 (0.015, 0.03%) was observed. Thus the effect of H2O2 on biologically relevant extracellular matrices for the formation of collagen scaffolds was shown.

Gelatin methacryloyl (GelMA) is a polymeric derivative of gelatin (Gel) widely used in tissue engineering, as well as in wound dressing and drug delivery field. This work offers an alternative and convenient method for the synthesis and characterization of GelMA. In particular, the methacrylation was carried out under homogeneous single-phase reaction conditions, which allows of obtaining a good reproducibility of the derivatization degree of the polymer in a simple and fast way. The experimental conditions were adequately optimized to have the methacrylation of only e-amino groups of lysine and hydroxylysine, as confirmed by FT-IR and 1H NMR analysis. The degree of derivatization of GelMA was determined using an internal standard applied to 1H NMR analysis. Based on these measurements, it was found that the optimized reaction proceeds to complete functionalization of e-amino groups of lysine and hydroxylysine. The effect of the reaction conditions on the rheological properties of GelMA was investigated through temperature sweep experiments and compared with those of the polymer obtained under traditional heterogeneous reaction conditions. A significant difference was found in the gelation temperature of GelMA synthesized following the two different procedures. In particular, the proposed single-phase synthetic protocol gave a polymer which behaves like a solution when used at 10% w/v concentration and even at temperatures as low as 5 °C, whereas the polymer obtained with the classic biphasic procedure behaves like a gel within the same range of temperatures and when used at the same concentration. These results are particularly interesting as they could open GelMA to new applications particularly in the biomedical field.

The diagenesis of a bone in the postmortem period causes an identifiable deterioration in histology. This degradation is characterized by a collagenous alteration, which can be observed very early. In order to develop a method for determining a postmortem interval for medico-legal use, two ribs collected from six human bodies were studied prospectively over 2 years. Each bone was studied after staining with Sirius red to demonstrate the degradation of collagen as a function of time. This study demonstrated a time-based bone alteration characterized by the architectural degradation of the lamellar bone, without any microbial influence in this postmortem period. The staining was carried out by using Sirius red and correlated this alteration with a collagenic degradation by chemical hydrolysis owing to the affinity of this dye to the amino acids lysine, hydroxylysine, and arginine. Our work asserts that human bone samples that were studied in a controlled environment and analyzed for 24 months underwent a diagenetic trajectory whose main element was collagen hydrolysis.

The low-molecular-weight (LMW) peptides derived from collagen have shown a potential for various nutritional and pharmaceutical applications. However, production of LMW peptides from vertebrate collagen remains a challenge. Herein, we report a new method to produce LMW collagen peptides using pepsin pretreatment that removed cross-linked telopeptides in collagen molecules. After the pretreatment, the proportion of LMW collagen peptides (1.4 kDa) that were obtained from pepsin-soluble collagen increased to 32.59% compared to heat-soluble collagen peptides (16.10%). Fourier transform infrared spectroscopy results indicated that telopeptide cleavage retained the triple-helical conformation of collagen. Liquid chromatography-tandem mass spectrometry analysis suggested that Gly-X-Y (X is often proline, while Y is either hydroxyproline or hydroxylysine) repeats were not the main factors that hindered the enzymatic hydrolysis of collagen molecules. However, cross-link quantification demonstrated that trivalent cross-links that included pyridinolines and pyrroles were the primary obstacles to producing small peptides from collagen of spent hens. This study demonstrated for the first time that removing cross-linked telopeptides could enhance the production of LMW peptides from spent hen collagen, which is also of interest to manufacturers who produce LMW collagen peptides from other vertebrate animals, such as bovids and porcids.

LOXL2 catalyzes the oxidative deamination of ε-amines of lysine and hydroxylysine residues within collagen and elastin, generating reactive aldehydes (allysine). Condensation with other allysines or lysines drives the formation of inter- and intramolecular cross-linkages, a process critical for the remodeling of the ECM. Dysregulation of this process can lead to fibrosis, and LOXL2 is known to be upregulated in fibrotic tissue. Small-molecules that directly inhibit LOXL2 catalytic activity represent a useful option for the treatment of fibrosis. Herein, we describe optimization of an initial hit 2, resulting in identification of racemic-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone 28, a potent irreversible inhibitor of LOXL2 that is highly selective over LOX and other amine oxidases. Oral administration of 28 significantly reduced fibrosis in a 14-day mouse lung bleomycin model. The (R,R)-enantiomer 43 (PAT-1251) was selected as the clinical compound which has progressed into healthy volunteer Phase 1 trials, making it the "first-in-class" small-molecule LOXL2 inhibitor to enter clinical development.

Osteogenesis imperfecta (OI), also known as brittle bone disease, displays a spectrum of clinical severity from mild (OI type I) to severe early lethality (OI type II), with clinical features including low bone mass, fractures, and deformities. Mutations in the FK506 Binding Protein 10 (FKBP10), gene encoding the 65-kDa protein FKBP65, cause a recessive form of OI and Bruck syndrome, the latter being characterized by joint contractures in addition to low bone mass. We previously showed that Fkbp10 expression is limited to bone, tendon, and ligaments in postnatal tissues. Furthermore, in both patients and Fkbp10 knockout mice, collagen telopeptide hydroxylysine crosslinking is dramatically reduced. To further characterize the bone specific contributions of Fkbp10, we conditionally ablated FKBP65 in Fkbp10fl/fl mice (Mus musculus; C57BL/6) using the osteoblast-specific Col1a1 2.3-kb Cre recombinase. Using μCT, histomorphometry and quantitative backscattered electron imaging, we found minimal alterations in the quantity of bone and no differences in the degree of bone matrix mineralization in this model. However, mass spectroscopy (MS) of bone collagen demonstrated a decrease in mature, hydroxylysine-aldehyde crosslinking. Furthermore, bone of mutant mice exhibits a reduction in mineral-to-matrix ratio and in crystal size as shown by Raman spectroscopy and small-angle X-ray scattering, respectively. Importantly, abnormalities in bone quality were associated with impaired bone biomechanical strength in mutant femurs compared with those of wild-type littermates. Taken together, these data suggest that the altered collagen crosslinking through Fkbp10 ablation in osteoblasts primarily leads to a qualitative defect in the skeleton. © 2017 American Society for Bone and Mineral Research.

Objectives The clinical, biochemical and genetic findings in two Slovak patients with glutaric aciduria type I (GAI) are presented. Background GAI is a rare autosomal recessive neuro-metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase, which is involved in the catabolic pathways of lysine, hydroxylysine and tryptophan. This enzymatic defect gives rise to elevated levels of glutaric acid (GA), 3-hydroxyglutaric acid (3-OH-GA) and glutarylcarnitine (C5DC) in body fluids. Methods Biochemical and molecular-genetic tests were performed. Urinary organic acids were analysed by Gas Chromatography/Mass Spectrometry (GC/MS) and the entire coding region of the GCDH gene, including flanking parts, was sequenced. Results We found the presence of typical metabolic profile and novel causal pathogenic variants in both GAI patients. Conclusion We present the first report of two Slovak patients with GAI, which differed in the clinical and biochemical phenotype significantly. They were diagnosed by two distinct approaches - selective and newborn screening. Their diagnosis was complexly confirmed by biochemical and later on molecular-genetic examinations. Though we agreed with a thesis that early diagnostics might positively influenced patient's health outcome, contradictory facts should be considered. Supposed extremely low prevalence of GAI patients in the general population and/or the existence of asymptomatic individuals with a questionable benefit of the applied therapeutic intervention for them lead to doubts whether the inclusion of disease into the newborn screening programme is justified well enough (Tab. 1, Fig. 3, Ref. 41).

Diabetic bone disease is a complication of type I and type II diabetes, both of which are increasing in the United States and elsewhere. Increased hip and foot fracture rates do not correlate well with changes in bone mineral density (BMD), whereas studies support the importance of collagen structure to bone strength. Extracellular lysyl oxidase (LOX) catalyzes the oxidative deamination of hydroxylysine and lysine residues in collagens resulting in aldehydes that subsequently form critically important biosynthetic crosslinks that stabilize functional collagens. Although LOX‐dependent biosynthetic crosslinks in bone collagen are deficient in diabetic bone, the expression and regulation of bone LOXs in diabetes have not been comprehensively studied. Here, we found that LOX is profoundly downregulated in bone in diabetes. Moreover, we have identified a novel metabolic regulatory relationship that is dysregulated in diabetes using mouse models. Data indicate that the incretin (gastric hormone) known as glucose‐dependent insulinotropic polypeptide (GIP) that is anabolic to osteoblasts strongly upregulates LOX, and that this regulation is disrupted in the streptozotocin‐induced model of diabetes in mice. In vivo and in vitro studies support that diabetes results in elevated circulating peripheral dopamine, likely also derived from the gut, and is responsible for blocking GIP signaling and LOX levels in osteoblasts. Moreover, peripheral administration of the dopamine D2 receptor antagonist amisulpride to diabetic mice restored trabecular bone structure to near normal and partially reversed downregulation of LOX. Taken together our data identifies a novel metabolic relationship between the gut‐derived hormone GIP and bone‐derived LOX, and points to the importance of LOX dysregulation in the pathology of diabetic bone disease. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

In a screen for organogenesis defects in N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we discovered a line carrying a mutation in Colgalt1 [collagen beta(1-O)galactosyltransferase type 1], which is required for proper galactosylation of hydroxylysine residues in a number of collagens. Colgalt1 mutant embryos have not been previously characterized; here, we show that they exhibit skeletal and muscular defects. Analysis of mutant-derived embryonic fibroblasts reveals that COLGALT1 acts on collagen IV and VI, and, while collagen VI appears stable and its secretion is not affected, collagen IV accumulates inside of cells and within the extracellular matrix, possibly due to instability and increased degradation. We also generated mutant zebrafish that do not express the duplicated orthologs of mammalian Colgalt1. The double-homozygote mutants have muscle defects; they are viable through the larvae stage but do not survive to 10 days post-fertilization. We hypothesize that the Colgalt1 mutant could serve as a model of a human connective tissue disorder and/or congenital muscular dystrophy or myopathy.

Lysyl oxidase-generated intermolecular cross-links are essential for the tensile strength of collagen fibrils. Two cross-linking pathways can be defined, one based on telopeptide lysine aldehydes and another on telopeptide hydroxylysine aldehydes. Since the 1970s it has been accepted that the mature cross-linking structures on the lysine aldehyde pathway, which dominates in skin and cornea, incorporate histidine residues. Here, using a range of MS-based methods, we re-examined this conclusion and found that telopeptide aldol dimerization is the primary mechanism for stable cross-link formation. The C-telopeptide aldol dimers formed labile addition products with glucosylgalactosyl hydroxylysine at α1(I)K87 in adjacent collagen molecules that resisted borohydride reduction and after acid hydrolysis produced histidinohydroxylysinonorleucine (HHL), but only from species with a histidine in their α1(I) C-telopeptide sequence. Peptide MS analyses and the lack of HHL formation in rat and mouse skin, species that lack an α1(I) C-telopeptide histidine, revealed that HHL is a laboratory artifact rather than a natural cross-linking structure. Our experimental results also establish that histidinohydroxymerodesmosine is produced by borohydride reduction of N-telopeptide allysine aldol dimers in aldimine intermolecular linkage to nonglycosylated α1(I) K930. Borohydride reduction of the aldimine promotes an accompanying base-catalyzed Michael addition of α1(I) H932 imidazole to the α,β-unsaturated aldol. These aldehydes are intramolecular at the N terminus but at the C terminus they can be both intramolecular and intermolecular according to present and earlier findings.

Solid-state nuclear magnetic resonance spectroscopy (ssNMR) is an emerging technique in structural methods of studying collagen proteins, capable of identifying features on an atomic length scale in tissues and protein samples without extensive extraction or purification. Hydroxylation is a key posttranslational modification of collagen that gives rise to distinctive signals in the ssNMR spectrum of collagen proteins. Here we outline the type of information that ssNMR can provide and describe the procedures involved in a ssNMR structural study, with particular focus on using dynamic nuclear polarization to enhance sensitivity for detecting hydroxylysine residues by ssNMR.

Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor lysyl hydroxylase (LH) activity, and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.

Collagen β (1-O) galactosyltransferase 1 (GLT25D1) has been reported to transfer galactose to hydroxylysine residues via β (1-O) linkages in collagen. However, the role of Glt25d1 in liver fibrogenesis is still unknow. Recently, we generated a Glt25d1 knockout mouse to elucidate the role of Glt25d1 in vivo. However, we found that complete deletion of the Glt25d1 gene resulted in embryonic lethality at E11.5. Histopathological analysis revealed that dysplasia in Glt25d1-/- labyrinth with defects of the vascular network. Immunohistochemical showed that the decrease in proliferation of Glt25d1-/- liver and the developing central nervous system (CNS). The role of Glt25d1 in liver fibrogenesis was explored by Glt25d1+/- mice. Glt25d1+/- mice and wild-type (WT) mice were injected intraperitoneally with the same dose of CCl4. The higher level of serum alanine aminotransferase was observed in Glt25d1+/- mice. Reverse transcription-quantitative polymerase chainreaction demonstrated that the mRNA expression levels of the inflammatory cytokines such as, Tnf-α, Cxcl-1 and Mcp-1, showed a significantly increase in CCl4-treated Glt25d1+/- mice. Collagen-I, collagen-III and α-SMA transcripts accumulation was markedly increased in the Glt25d1+/- mice. However, Masson's trichrome staining revealed a trend to decrease in the ECM proteins deposition of Glt25d1+/- liver. Immunohistochemistry and Western blots revealed that the protein expression of Collagen-III was reduced and a trend to a decrease in collagen-I was observed in the Glt25d1+/- liver compared with those of WT mice. Our results demonstrate that Glt25d1 knockout results in embryonic lethality and down-regulation of Glt25d1 may inhibit collagen secretion during liver fibrogenesis.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

Glutaric aciduria type 1 is an autosomal recessive disorder caused by mutations in GCDH gene on chromosome 19 leading to the deficiency of glutaryl-CoA dehydrogenase which causes an abnormal metabolism of lysine, hydroxylysine and tryptophan with resultant accumulation of glutaric acid and 3-hydroxy glutaric acid. Usual presentations include macrocephaly with recurrent dystonic episodes, along with developmental regression. The diagnosis is based on characteristic magnetic resonance imaging finding of widening of sylvian fissures and urinary tandem mass spectroscopic analysis of excess glutarylcarnitine and hydroxyglutaric acid. Management includes lysine-free diet and carnitine supplementation.

Background This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Methods Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. Results It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%–24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. Conclusions The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. General significance The study for the first time shows that prolyl hydroxylation is highly regulated in elastin.

To determine the nutritional value of domestic tomatoes, the levels of amino acids, amino acid metabolites, and the bioactive compound γ-aminobutyric-acid (GABA) were analyzed in three domestic tomato varieties (Rafito, Momotaro, and Medison). Eighteen free amino acids were found, and total free amino acid content was 3,810.21～4,594.56 mg/100 g (dry weight). L-glutamic acid (L-Glu) was the most abundant amino acid, ranging from 1,866.60 mg/100 g for Momotaro to 2,417.45 mg/100 g for Medison. The next most abundant amino acids were L-glutamine (L-Gln) and L-aspartic acid (L-Asp). The three tomato varieties had a good balance of all the essential amino acids except tryptophan. Total essential amino acid content was 274.26～472.71 mg/100 g (dry weight). The following amino acid metabolites were found: L-carnitine (L-Car), hydroxylysine (Hyl), o-phosphoethanolamine (o-Pea), phosphoserine (p-Ser), β-alanine (β-Ala), N-methyl-histidine (Me-His), ethanolamine (EtNH2),and L-citrulline(L-Cit). Large quantities of GABA were found in all three varieties: 666.95-868.48 mg/100g (dry weight). These results support the use of these tomato varieties as nutritious food materials.

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability.

We investigated alternatives to commonly used biomarkers of exercise-induced tissue damage. Over 5 days following two bouts of 100 drop-to-vertical jumps (inter-bout rest period of 3 weeks), myosin heavy chain 1, hydroxylysine (HYL), hydroxyproline (HYP), spermine (SPM) and spermine synthase (SMS) were measured in the serum of 10 participants. HYL significantly increased from 5.92 ± 1.49 ng/mL to 6.48 ± 1.47 ng/mL at 24 h. A similar trend was observed for bout 2, but without reaching significance. SPM significantly increased only after bout 1 from 0.96 ± 0.19 ng/mL at pretest to a peak level of 1.12 ± 0.26 ng/mL at 24 h, while B2 increments remained non-significant. Myosin heavy chain 1, HYP and SMS values remained below the detection limit of the applied enzyme-linked immunosorbent assay (ELISA) kit. Though HYL and SM increased after the intervention, both markers showed a large standard deviation (SD) combined with small increments. Therefore, none of the investigated biomarkers provides a meaningful alternative to commonly used damage markers.

d -Glucose derived synthon 4 was successfully utilized for the synthesis of (2S,3R)-β-hydroxyornithine 1, (2S,3R)-β-hydroxylysine 2 and tetrahydroazepine core of balanol 3.

// Wang-Ren Qiu 1, 2 , Bi-Qian Sun 1 , Xuan Xiao 1, 3 , Zhao-Chun Xu 1 , Kuo-Chen Chou 3, 4, 5 1 Computer Department, Jingdezhen Ceramic Institute, Jingdezhen, China 2 Department of Computer Science and Bond Life Science Center, University of Missouri, Columbia, MO, USA 3 Gordon Life Science Institute, Boston, MA, USA 4 Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia 5 Center of Bioinformatics, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan, China Correspondence to: Xuan Xiao, email: xxiao@gordonlifescience.org Keywords: PTMs, hydroxyproline, hydroxylysine, sequence-coupling model, general PseAAC Received: April 20, 2016 Accepted: May 29, 2016 Published: June 14, 2016 ABSTRACT Protein hydroxylation is a posttranslational modification (PTM), in which a CH group in Pro (P) or Lys (K) residue has been converted into a COH group, or a hydroxyl group (-OH) is converted into an organic compound. Closely associated with cellular signaling activities, this type of PTM is also involved in some major diseases, such as stomach cancer and lung cancer. Therefore, from the angles of both basic research and drug development, we are facing a challenging problem: for an uncharacterized protein sequence containing many residues of P or K, which ones can be hydroxylated, and which ones cannot? With the explosive growth of protein sequences in the post-genomic age, the problem has become even more urgent. To address such a problem, we have developed a predictor called iHyd-PseCp by incorporating the sequence-coupled information into the general pseudo amino acid composition (PseAAC) and introducing the “Random Forest” algorithm to operate the calculation. Rigorous jackknife tests indicated that the new predictor remarkably outperformed the existing state-of-the-art prediction method for the same purpose. For the convenience of most experimental scientists, a user-friendly web-server for iHyd-PseCp has been established at http://www.jci-bioinfo.cn/iHyd-PseCp , by which users can easily obtain their desired results without the need to go through the complicated mathematical equations involved.

Hydroxylation of proline or lysine residues in proteins is a common post-translational modification event, and such modifications are found in many physiological and pathological processes. Nonetheless, the exact molecular mechanism of hydroxylation remains under investigation. Because experimental identification of hydroxylation is time-consuming and expensive, bioinformatics tools with high accuracy represent desirable alternatives for large-scale rapid identification of protein hydroxylation sites. In view of this, we developed a supporter vector machine-based tool, OH-PRED, for the prediction of protein hydroxylation sites using the adapted normal distribution bi-profile Bayes feature extraction in combination with the physicochemical property indexes of the amino acids. In a jackknife cross validation, OH-PRED yields an accuracy of 91.88% and a Matthew's correlation coefficient (MCC) of 0.838 for the prediction of hydroxyproline sites, and yields an accuracy of 97.42% and a MCC of 0.949 for the prediction of hydroxylysine sites. These results demonstrate that OH-PRED increased significantly the prediction accuracy of hydroxyproline and hydroxylysine sites by 7.37 and 14.09%, respectively, when compared with the latest predictor PredHydroxy. In independent tests, OH-PRED also outperforms previously published methods.