Sulfilimine bond formation in collagen IV.

The collagen IV network plays a crucial role in providing structural support and mechanical integrity to the basement membrane and surrounding tissues. A key aspect of this network is the formation of intra- and inter-collagen fibril crosslinks. One particular crosslink, an inter-residue sulfilimine bond, has been found, so far, to be unique to collagen IV. More specifically, these crosslinks are primarily formed between methionine and lysine or hydroxylysine residues and can occur within a single collagen fibril or between different collagen fibrils. Due to its significance as the major crosslink in the collagen IV network, the sulfilimine bond plays critical roles in tissue development and various human diseases. While the proposed reaction mechanism for sulfilimine bond formation is supported by experimental evidence, the precise nature of this bond remained uncertain until computational studies were conducted. The process involves the reaction of hypohalous acids (e.g., HOBr, HOCl), produced by a peroxidasin enzyme in the basement membrane, with the sidechain sulfur of methionine or sidechain nitrogen of lysine/hydroxylysine residues in collagen IV, to form halosulfonium or haloamine intermediates, respectively. The halosulfonium/haloamine then reacts with the sidechain amine/sulfide of the lysine (or hydroxylysine) or methionine respectively, eventually resulting in the formation of the sulfilimine (MetSNLys/Hyl) crosslink. The sulfilimine product formed not only plays a crucial role in physiological processes but also finds applications in various industrial and pharmaceutical contexts. In this review, we provide a comprehensive summary of existing studies, including our own research, aimed at understanding the reaction mechanism, protonation states, characteristic nature, and dynamic behavior of the sulfilimine bond in collagen IV. The goal is to offer readers an overview of this critically important biochemical bond.

Differences in serum metabolome profile explain individual variation in growth performance of young goats.

High growth rates and body weight are important traits of young dairy goats that can shorten generation intervals, improve animal performance, and increase economic benefits. In the present study, ninety-nine, 6-month-old, female goats were fed with the same diet and kept under the same management condition. The ten goats with highest average daily gain (ADG, HADG, 135.27 ± 4.59 g/d) and ten goats with lowest ADG (LADG, 87.74 ± 3.13 g/d) were selected to identify the key serum metabolites associated with ADG, and to investigate the relationships of serum metabolome profiles with digestive tract microbiota. The results showed that a total of 125 serum metabolites were significantly different between HADG and LADG. Of these, 43 serum metabolites were significantly higher levels in HADG, including D-ornithine, l-glutamine, L-histidine, carnosine, LysoPC (16:1(9Z)/0:0), DCTP and hydroxylysine, while, 82 serum metabolites were significantly higher levels in LADG, including P-salicylic acid and deoxycholic acid 3-glucuronide. Pathway analysis indicated that these different metabolites were mainly involved in amino acid and lipid metabolism. Furthermore, Spearman's rank correlation analysis revealed that these differential serum metabolites were correlated with ADG and ADG-related bacteria. Notably, serum hydroxylysine and L-histidine could be used as biomarkers for distinguishing HADG and LADG goats, with an accuracy of >92.0%. SIGNIFICANCE: Our study confirms that individual microbiota and metabolic differences contribute to the variations of growth rate in young goats. Some serum metabolites may be useful in improving the growth performance of young goats, which provides directions for developing further nutritional regulation in the goat industry to achieve healthy feeding and efficiency enhancement.

Identification of Regulatory Molecular "Hot Spots" for LH/PLOD Collagen Glycosyltransferase Activity.

Hydroxylysine glycosylations are post-translational modifications (PTMs) essential for the maturation and homeostasis of fibrillar and non-fibrillar collagen molecules. The multifunctional collagen lysyl hydroxylase 3 (LH3/PLOD3) and the collagen galactosyltransferase GLT25D1 are the human enzymes that have been identified as being responsible for the glycosylation of collagen lysines, although a precise description of the contribution of each enzyme to these essential PTMs has not yet been provided in the literature. LH3/PLOD3 is thought to be capable of performing two chemically distinct collagen glycosyltransferase reactions using the same catalytic site: an inverting beta-1,O-galactosylation of hydroxylysines (Gal-T) and a retaining alpha-1,2-glucosylation of galactosyl hydroxylysines (Glc-T). In this work, we have combined indirect luminescence-based assays with direct mass spectrometry-based assays and molecular structure studies to demonstrate that LH3/PLOD3 only has Glc-T activity and that GLT25D1 only has Gal-T activity. Structure-guided mutagenesis confirmed that the Glc-T activity is defined by key residues in the first-shell environment of the glycosyltransferase catalytic site as well as by long-range contributions from residues within the same glycosyltransferase (GT) domain. By solving the molecular structures and characterizing the interactions and solving the molecular structures of human LH3/PLOD3 in complex with different UDP-sugar analogs, we show how these studies could provide insights for LH3/PLOD3 glycosyltransferase inhibitor development. Collectively, our data provide new tools for the direct investigation of collagen hydroxylysine PTMs and a comprehensive overview of the complex network of shapes, charges, and interactions that enable LH3/PLOD3 glycosyltransferase activities, expanding the molecular framework and facilitating an improved understanding and manipulation of glycosyltransferase functions in biomedical applications.

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.

Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition.

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

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.

N-Succinyltransferase Encoded by a Cryptic Siderophore Biosynthesis Gene Cluster in Streptomyces Modifies Structurally Distinct Antibiotics.

The antibiotic desertomycin A and its previously undescribed inactive N-succinylated analogue, desertomycin X, were isolated from Streptomyces sp. strain YIM 121038. Genome sequencing and analysis readily identified the desertomycin biosynthetic gene cluster (BGC), which lacked genes encoding acyltransferases that would account for desertomycin X formation. Scouting the genome for putative N-acyltransferase genes led to the identification of a candidate within a cryptic siderophore BGC (csb) encoding a putative homologue of the N6'-hydroxylysine acetyltransferase IucB. Expression of the codon-optimized gene designated csbC in Escherichia coli yielded the recombinant protein that was able to N-succinylate desertomycin A as well as several other structurally distinct antibiotics harboring amino groups. Some antibiotics were rendered antibiotically inactive due to the CsbC-catalyzed succinylation in vitro. Unlike many known N-acyltransferases involved in antibiotic resistance, CsbC could not efficiently acetylate the same antibiotics. When expressed in E. coli, CsbC provided low-level resistance to kanamycin and ampicillin, suggesting that it may play a role in antibiotic resistance in natural habitats, where the concentration of antibiotics is usually low. IMPORTANCE In their natural habitats, bacteria encounter a plethora of organic compounds, some of which may be represented by antibiotics produced by certain members of the microbial community. A number of antibiotic resistance mechanisms have been described, including those specified by distinct genes encoding proteins that degrade, modify, or expel antibiotics. In this study, we report identification and characterization of an enzyme apparently involved in the biosynthesis of a siderophore, but also having the ability of modify and thereby inactivate a wide variety of structurally diverse antibiotics. This discovery sheds light on additional capabilities of bacteria to withstand antibiotic treatment and suggests that enzymes involved in secondary metabolism may have an additional function in the natural environment.

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.

Metabolic characterization of amniotic fluid of fetuses with isolated choroid plexus cyst.

OBJECTIVES: To investigate the amino acid (AA)-related metabolic characteristics of amniotic fluid (AF) obtained by ultrasound-guided amniocentesis from fetuses with isolated choroid plexus cysts of the central nervous system. METHODS: Ultrasound-guided amniocentesis was performed on 17 fetuses with isolated choroid plexus cysts (ICPCs) and 17 normal fetuses. The AF samples from normal pregnancies were matched with the case samples in a 1:1 ratio based upon gestational age. The AF samples from the 34 fetuses were analyzed by liquid chromatography-mass spectrometry (LC-MS). Then, the peak areas of the metabolites were analyzed by principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and univariate statistical analysis. RESULTS: This study ultimately identified 31 AAs. Seven differentially abundant AAs were screened out, including citrulline, ethanolamine, aspartic acid, valine, 5-hydroxylysine, proline, and isoleucine (p-value<0.05). A total of 4 metabolic pathways were significantly altered in the ICPC group: valine, leucine and isoleucine biosynthesis; valine, leucine and isoleucine degradation; pantothenate and coenzyme A (CoA) biosynthesis; and arginine biosynthesis. CONCLUSIONS: The results of this study indicate that fetuses with ICPC have disrupted levels of citrulline, ethanolamine, aspartic acid, valine, 5-hydroxylysine, proline, and isoleucine, which may ultimately affect fetal glucose and lipid metabolism.

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.