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.

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.

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.

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.

Glycosylation of Type I Collagen.

Fibrillar type I collagen is the most abundant structural protein in most tissues and organs. One of the unique and functionally important characteristics of collagen is sequential posttranslational modifications of lysine (Lys) residues. In the endoplasmic reticulum, hydroxylation of specific Lys occurs producing 5-hydroxylysine (Hyl). Then, to the 5-hydroxyl group of Hyl, a single galactose unit can be attached to form galactosyl-Hyl (Gal-Hyl) and further glucose can be added to Gal-Hyl to form glucosylgalactosyl-Hyl (GlcGal-Hyl). These are the only two O-linked glycosides found in mature type I collagen. It has been shown that this modification is critically involved in a number of biological and pathological processes likely through its regulatory roles in collagen fibrillogenesis, intermolecular cross-linking, and collagen-cell interaction. Recently, with the advances in molecular/cell biology and analytical chemistry, the molecular mechanisms of collagen glycosylation have been gradually deciphered, and the type and extent of glycosylation at the specific molecular loci can now be quantitatively analyzed. In this chapter, we describe quantitative analysis of collagen glycosylation by high-performance liquid chromatography (HPLC) and semiquantitative, site-specific analysis by HPLC-tandem mass spectrometry.

Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.

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,ß-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.

A Hybrid Deep Learning Model for Predicting Protein Hydroxylation Sites.

Protein hydroxylation is one type of post-translational modifications (PTMs) playing critical roles in human diseases. It is known that protein sequence contains many uncharacterized residues of proline and lysine. The question that needs to be answered is: which residue can be hydroxylated, and which one cannot. The answer will not only help understand the mechanism of hydroxylation but can also benefit the development of new drugs. In this paper, we proposed a novel approach for predicting hydroxylation using a hybrid deep learning model integrating the convolutional neural network (CNN) and long short-term memory network (LSTM). We employed a pseudo amino acid composition (PseAAC) method to construct valid benchmark datasets based on a sliding window strategy and used the position-specific scoring matrix (PSSM) to represent samples as inputs to the deep learning model. In addition, we compared our method with popular predictors including CNN, iHyd-PseAAC, and iHyd-PseCp. The results for 5-fold cross-validations all demonstrated that our method significantly outperforms the other methods in prediction accuracy.

Glycation of type I collagen selectively targets the same helical domain lysine sites as lysyl oxidase-mediated cross-linking.

Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal cross-linking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties.

Fabrication of nanofibrous microcarriers mimicking extracellular matrix for functional microtissue formation and cartilage regeneration.

Cartilage has rather limited capacities for self-repair and regeneration. To repair complexly shaped cartilage tissue defects, we propose the application of microtissues fabricated from bone marrow-derived mesenchymal stem cells (BMSCs) cultured in natural bionic nanofibrous microcarriers (NF-MCs). The NF-MCs were structurally and functionally designed to mimic natural extracellular matrix (ECM) by crosslinking dialdehyde bacterial cellulose (DBC) with DL-allo-hydroxylysine (DHYL) and complexing chitosan (CS) with DHYL through electrostatic interactions. The orthogonal design allows for fine tuning of fiber diameter, pore size, porosity, mechanical properties, and biodegradation rate of the NF-MC. BMSCs cultured in NF-MCs showed improved proliferation compared with those cultured in chitosan microcarriers (CS-MCs). After three-week culture under microgravity conditions, functional cartilage microtissues were generated. When implanted into a knee articular cartilage defect in mice, the microtissue showed superior in vivo cartilage repair as characterized by cell tracking, histology, micro CT image, and gait analysis. Versatile in natural biopolymer design and biomimetic in nanofibrous component embedded in macroporous microcarriers, these injectable NC-MCs demonstrate to be effective carriers for cell proliferation and differentiation. Furthermore, the functional microtissues also show their prospect in repair of cartilage tissue, and suggest their potential for other tissues in general.

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.

Focus on O-phosphohydroxylysine, O-phosphohydroxyproline, N 1-phosphotryptophan and S-phosphocysteine.

The synthesis and chemistry of the lesser-known phosphoamino acids, O-phosphohydroxylysine, O-phosphohydroxyproline, N 1-phosphotryptophan and S-phosphocysteine are described in detail. In addition, where anything at all is known, the biological synthesis, occurrence and functions of these phosphoamino acids are described. Of these phosphoamino acids, only N 1-phosphotryptophan has not been reported to occur in proteins; however, apart from the roles of S-phosphocysteine in the sugar transporter component (EII) and in catalysis by protein phosphotyrosine phosphatase, little is currently known about the biological roles of the phosphoamino acids when they occur as post-translational modifications.

iHyd-PseCp: Identify hydroxyproline and hydroxylysine in proteins by incorporating sequence-coupled effects into general PseAAC.

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.

The collaggrecan: Synthesis and visualization of an artificial proteoglycan.

An artificial aggrecan-like proteoglycan has been designed and synthesized in vitro. At variance with natural proteoglycans, whose glycosaminoglycan chains are always O-linked via a tetrasaccharide bridge to the serine residues of a specific protein core, the present structure consists of chondroitin-6-sulfate chains directly bound to the lysine and hydroxylysine residues of a collagen molecule backbone. The resulting macromolecule has been characterized by histochemistry, atomic force microscopy and FTIR. The number of variables involved (e.g., length and type of the collagen backbone, glycosaminoglycan species, sulfation type and pattern, molecular weight, number and length of side chains, etc.) makes possible to conceive an almost endless variety of artificial proteoglycans, each precisely tailored to a specific functional role. In addition to their use as biomaterials, glycated collagens interact with cells in complex ways and a previous study has already shown the ability of a glycated collagen to redirect fibroblastoma cells from proliferation to differentiation. The research is still underway.

Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies.

Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MS(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified.

2-Oxazoline formation for selective chemical labeling of 5-hydroxylysine.

Hydroxylation of lysine, one of posttranslational modifications of proteins, generates 5-hydroxylysine (Koh) and plays a crucial role in regulating protein functions in cellular activity. We have developed a chemical labeling method of Koh. The 1,2-aminoalcohol moiety of Koh in synthetic peptide sequences was trapped by an alkyne-containing benzimidate to form a 2-oxazoline ring. An additional ammonia treatment process removed the undesirable amidine residue formed between benzimidate and lysine. During the ammonia treatment, the oxazoline residue formed at Koh mainly remained intact, and the ring opening to the amide form was observed for only part of oxazoline, indicating that the chemical labeling is amino acid selective. Azide-substituted biotin or fluorescent dye was attached to the peptide through Huisgen cycloaddition at Koh and converted into an alkyne-labeled oxazoline form. The Koh-labeling assay could provide a platform to enhance proteomic research of lysine hydroxylation.

Facile synthesis of 5-hydroxy-L-lysine from D-galactose as a chiral-precursor.

A concise synthesis of (2S,5R) and (2S,5S)-5-hydroxy-lysine was achieved by utilizing D-galactose as a chiral-precursor with stereo retention. This synthetic strategy showcased the potential of utilizing carbohydrates as starting materials to prepare amino acids. Using the diazido intermediate, the derived ß-D-galactopyranosyl and α-D-glucopyranosyl-(1→2)-ß-D-galactosyl moieties were synthesized.

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.

iHyd-PseAAC: predicting hydroxyproline and hydroxylysine in proteins by incorporating dipeptide position-specific propensity into pseudo amino acid composition.

Post-translational modifications (PTMs) play crucial roles in various cell functions and biological processes. Protein hydroxylation is one type of PTM that usually occurs at the sites of proline and lysine. Given an uncharacterized protein sequence, which site of its Pro (or Lys) can be hydroxylated and which site cannot? This is a challenging problem, not only for in-depth understanding of the hydroxylation mechanism, but also for drug development, because protein hydroxylation is closely relevant to major diseases, such as stomach and lung cancers. With the avalanche of protein sequences generated in the post-genomic age, it is highly desired to develop computational methods to address this problem. In view of this, a new predictor called "iHyd-PseAAC" (identify hydroxylation by pseudo amino acid composition) was proposed by incorporating the dipeptide position-specific propensity into the general form of pseudo amino acid composition. It was demonstrated by rigorous cross-validation tests on stringent benchmark datasets that the new predictor is quite promising and may become a useful high throughput tool in this area. A user-friendly web-server for iHyd-PseAAC is accessible at http://app.aporc.org/iHyd-PseAAC/. Furthermore, for the convenience of the majority of experimental scientists, a step-by-step guide on how to use the web-server is given. Users can easily obtain their desired results by following these steps without the need of understanding the complicated mathematical equations presented in this paper just for its integrity.

A fluorogenic probe for recognizing 5-hydroxylysine inspired by serine/threonine ligation.

The 5-lysyl-hydroxylation has been found among protein post-translational modifications. In this report, we have devised a fluorescence turn-on probe which allows for selectively recognizing 5-hydroxylysine-containing peptides.

Synthesis of an azido precursor to (2S,5R)-5-hydroxylysine using an asymmetric organocatalytic chlorination/reduction sequence.

An efficient, robust, and scalable synthesis of an azido precursor to the modified amino acid (2S,5R)-5-hydroxylysine was developed on the basis of the use of a highly stereoselective organocatalytic α-chlorination-reduction protocol. The final Fmoc-protected (2S,5R)-6-azido-5-hydroxylysine derivative can be used in solid-phase peptide synthesis, providing access to proteins that contain large quantities of post-translationally modified lysine (e.g., collagens).