Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein.

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Effect of Dexamethasone on Adhesion of Human Neutrophils and Concomitant Secretion.

Neutrophils play a dual role in protecting the body. They are able to penetrate infected tissues and destroy pathogens there by releasing aggressive bactericidal substances. While into the surrounding tissues, the aggressive products secreted by neutrophils initiate development of inflammatory processes. Invasion of neutrophils into tissues is observed during the development of pneumonia in the patients with lung diseases of various etiologies, including acute respiratory distress syndrome caused by coronavirus disease. Synthetic corticosteroid hormone dexamethasone has a therapeutic effect in treatment of lung diseases, including reducing mortality in the patients with severe COVID-19. The acute (short-term) effect of dexamethasone on neutrophil adhesion to fibrinogen and concomitant secretion was studied. Dexamethasone did not affect either attachment of neutrophils to the substrate or their morphology. Production of reactive oxygen species (ROS) and nitric oxide (NO) by neutrophils during adhesion also did not change in the presence of dexamethasone. Dexamethasone stimulated release of metalloproteinases in addition to the proteins secreted by neutrophils during adhesion under control conditions, and selectively stimulated release of free amino acid hydroxylysine, a product of lysyl hydroxylase. Metalloproteinases play a key role and closely interact with lysyl hydroxylase in the processes of modification of the extracellular matrix. Therapeutic effect of dexamethasone could be associated with its ability to reorganize extracellular matrix in the tissues by changing composition of the neutrophil secretions, which could result in the improved gas exchange in the patients with severe lung diseases.

A basic study for the molecular imaging of dual-energy CT in diagnosing anterior cruciate ligament injury of knee joint.

BACKGROUND: Anterior cruciate ligament (ACL) injury is a common disease in clinical practice that seriously affects the daily life of patients. PURPOSE: To explore the molecular imaging basis of "diminution sign on dual-energy colour mapping" for the diagnosis of ACL injury by dual-energy computed tomography (DECT). MATERIAL AND METHODS: The hydroxylysine and hydroxyproline reagents were prepared in different concentrations. The grouping was shown as follows: a simple concentration change group of an amino acid (group 1/2); a mixed solution group with the concentration increasing synchronously (group 3); a mixed solution group with the concentration reverse increasing and decreasing (group 4); and a mixed solution group that fix one amino acid with increasing concentration of the other (group 5/6). The samples were scanned by DECT. The solution CT value and image signal-to-noise ratio were analyzed. RESULTS: In group 1/2, the brightness of the dual-energy color mapping of each test tube solution and the CT value increased with increasing the concentration of amino acid. In group 6, there was no significant change in the brightness and brilliance of the dual-energy color mapping and the CT value. The remaining three groups showed an increase in the brightness and brilliance of the dual-energy color mapping and the CT value, and this increase was positively associated with the hydroxylysine concentration. CONCLUSION: The dual-energy staining of the DECT imaging in "tendon" mode is related to hydroxylysine and hydroxyproline. Moreover, the degree of dual-energy color mapping is positively correlated with the change of CT value.

Efficient and scalable synthesis of 1,5-diamino-2-hydroxy-pentane from L-lysine via cascade catalysis using engineered Escherichia coli.

BACKGROUND: 1,5-Diamino-2-hydroxy-pentane (2-OH-PDA), as a new type of aliphatic amino alcohol, has potential applications in the pharmaceutical, chemical, and materials industries. Currently, 2-OH-PDA production has only been realized via pure enzyme catalysis from lysine hydroxylation and decarboxylation, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2-OH-PDA for industrial applications, but the substrate transport rate, appropriate catalytic environment (pH, temperature, ions) and separation method restrict its efficient synthesis. Here, a strategy was developed to produce 2-OH-PDA via an efficient, green and sustainable biosynthetic method on an industrial scale. RESULTS: In this study, an approach was created for efficient 2-OH-PDA production from L-lysine using engineered E. coli BL21 (DE3) cell catalysis by a two-stage hydroxylation and decarboxylation process. In the hydroxylation stage, strain B14 coexpressing L-lysine 3-hydroxylase K3H and the lysine transporter CadB-argT enhanced the biosynthesis of (2S,3S)-3-hydroxylysine (hydroxylysine) compared with strain B1 overexpressing K3H. The titre of hydroxylysine synthesized by B14 was 2.1 times higher than that synthesized by B1. Then, in the decarboxylation stage, CadA showed the highest hydroxylysine activity among the four decarboxylases investigated. Based on the results from three feeding strategies, L-lysine was employed to produce 110.5 g/L hydroxylysine, which was subsequently decarboxylated to generate a 2-OH-PDA titre of 80.5 g/L with 62.6% molar yield in a 5-L fermenter. In addition, 2-OH-PDA with 95.6% purity was obtained by solid-phase extraction. Thus, the proposed two-stage whole-cell biocatalysis approach is a green and effective method for producing 2-OH-PDA on an industrial scale. CONCLUSIONS: The whole-cell catalytic system showed a sufficiently high capability to convert lysine into 2-OH-PDA. Furthermore, the high titre of 2-OH-PDA is conducive to separation and possesses the prospect of industrial scale production by whole-cell catalysis.

Intervertebral disc degeneration in warmblood horses: Histological and biochemical characterization.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

Collagen hydroxylysine glycosylation: non-conventional substrates for atypical glycosyltransferase enzymes.

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.

Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein.

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.

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6.

JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.

Effect of hydroxylysine-O-glycosylation on the structure of type I collagen molecule: A computational study.

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.

Free urinary glycosylated hydroxylysine as an indicator of altered collagen degradation in the mucopolysaccharidoses.

Extracellular matrix (ECM) disruption is known to be an early pathological feature of the Mucopolysaccharidoses (MPS). Collagen is the main component of the ECM and its metabolism could act as a useful indicator of ECM disruption. We have measured the specific collagen breakdown products; urinary free hydroxylated (Lys-OH) and glycosylated hydroxylysines (Lys-O-Gal and Lys-O-GalGlc) in MPS patients using a tandem liquid chromatography tandem mass spectrometry assay. A pilot study cohort analysis indicated that concentrations of lysine and Lys-OH were raised significantly in MPS I (Hurler) disease patients. Lys-O-GalGlc was raised in MPS II and MPS VI patients and demonstrated a significant difference between MPS I Hurler and an MPS I Hurler-Scheie group. Further analysis determined an age association for glycosylated hydroxylysine in control samples similar to that observed for the glycosaminoglycans. Using defined age ranges and treatment naïve patient samples we confirmed an increase in glycosylated hydroxylysines in MPS I and in adult MPS IVA. We also looked at the ratio of Lys-O-Gal to Lys-O-GalGlc, an indicator of the source of collagen degradation, and noticed a significant change in the ratio for all pediatric MPS I, II, and IV patients, and a small significant increase in adult MPS IV. This indicated that the collagen degradation products were coming from a source other than bone such as cartilage or connective tissue. To see how specific the changes in glycosylated hydroxylysine were to MPS patients we also looked at levels in patients with other inherited metabolic disorders. MPS patients showed a trend towards increased glycosylated hydroxylysines and an elevated ratio compared to other metabolic disorders that included Battens disease, Fabry disease, Pyridoxine-dependent epilepsy (due to mutations in ALDH7A1), and Niemann Pick C disease.

iHyd-LysSite (EPSV): Identifying Hydroxylysine Sites in Protein Using Statistical Formulation by Extracting Enhanced Position and Sequence Variant Feature Technique.

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 cross-validation 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.

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation.

Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space.

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147-1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.

Prolyl hydroxylation in elastin is not random.

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.

Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction.

Hydroxylation via C-H bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available l-lysine with l-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two l-lysine 3S-hydroxylases and four l-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of l-lysine, yielding (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant l-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S,3S)-3-hydroxylysine was produced from 600 mM l-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S,4R)-4-hydroxylysine was produced from 300 mM l-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies.IMPORTANCE The present study identified six l-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of l-lysine hydroxylases was relatively narrow, we found that (2S,3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S,5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the l-arginine hydroxylase VioC also hydroxylated l-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of l-lysine into (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic l-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry.

Collagen IV is the main structural protein that provides a scaffold for assembly of basement membrane proteins. Posttranslational modifications such as hydroxylation of proline and lysine and glycosylation of lysine are essential for the functioning of collagen IV triple-helical molecules. These modifications are highly abundant posing a difficult challenge for in-depth characterization of collagen IV using conventional proteomics approaches. Herein, we implemented an integrated pipeline combining high-resolution mass spectrometry with different fragmentation techniques and an optimized bioinformatics workflow to study posttranslational modifications in mouse collagen IV. We achieved 82% sequence coverage for the α1 chain, mapping 39 glycosylated hydroxylysine, 148 4-hydroxyproline, and seven 3-hydroxyproline residues. Further, we employed our pipeline to map the modifications on human collagen IV and achieved 85% sequence coverage for the α1 chain, mapping 35 glycosylated hydroxylysine, 163 4-hydroxyproline, and 14 3-hydroxyproline residues. Although lysine glycosylation heterogeneity was observed in both mouse and human, 21 conserved sites were identified. Likewise, five 3-hydroxyproline residues were conserved between mouse and human, suggesting that these modification sites are important for collagen IV function. Collectively, these are the first comprehensive maps of hydroxylation and glycosylation sites in collagen IV, which lay the foundation for dissecting the key role of these modifications in health and disease.

Polyamines, myosin heavy chains, and collagen specific amino acids after a repeated bout of eccentric exercise.

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.

Glycosylation modulates melanoma cell α2ß1 and α3ß1 integrin interactions with type IV collagen.

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2ß1 and α3ß1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3ß1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2ß1 integrin interaction with α1(IV)382-393 but right in the middle of α3ß1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no ß-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.

Glycosylation and cross-linking in bone type I collagen.

Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998-23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen.