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.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

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.

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.

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease.

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.

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.

Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine, l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro.

Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-oxoglutarate dehydrogenase complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate dehydrogenase complex (hOADHc) from the final degradation pathway of l-lysine, l-hydroxylysine and l-tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (kcat/Km) were displayed by the two complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subcomplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both complexes produced superoxide/H2O2 from O2 in the reductive half reaction suggesting that hE1o, and hE1a (within their complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two complexes on the different metabolic pathways.

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.

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.

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen.

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼ 120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.

Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry.

Glycosylation is one of the most common post-translational modifications in proteins, existing in ~50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites.

Effect of 3D chondrocyte culturing conditions on the formation of extracellular matrix in cartilage tissue-engineering constructs.

We studied biochemical, morphological, and histological parameters of the extracellular matrix in scaffold-free tissue engineering chondrotransplants prepared from chondrocytes isolated from knee joint cartilage biopsy specimens of Haflinger horses (age 3.5-14 years) and in transplants prepared on the basis of commercial matrixes of Ethisorb® and Chondro-Gide(®). A total of 50 tissue-engineering constructs were designed and analyzed. Passage 2 cells populations were used. Mechanical stimulation during culturing of scaffold-free constructs considerably activated synthesis of the basic components of the cartilage matrix (proteoglycan concentration was 35% of the content in the native tissue, and the content of collagen-specific amino acids hydroxyproline and hydroxylysine attained 29.3 and 12.7%, respectively). The architecture of these cartilage constructs was morphologically and histologically similar to the native cartilage tissue. Insufficient support of the chondrogenesis by scaffold-based chondrotransplants and no differences between these constructs by the studied parameters were noted despite different chemical nature and structure of these scaffolds.

Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance.

Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846-8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.

Molecular identification of hydroxylysine kinase and of ammoniophospholyases acting on 5-phosphohydroxy-L-lysine and phosphoethanolamine.

The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40-50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-L-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-L-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-L-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines.

Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture.

Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.

Metabolite identification using automated comparison of high-resolution multistage mass spectral trees.

Multistage mass spectrometry (MS(n)) generating so-called spectral trees is a powerful tool in the annotation and structural elucidation of metabolites and is increasingly used in the area of accurate mass LC/MS-based metabolomics to identify unknown, but biologically relevant, compounds. As a consequence, there is a growing need for computational tools specifically designed for the processing and interpretation of MS(n) data. Here, we present a novel approach to represent and calculate the similarity between high-resolution mass spectral fragmentation trees. This approach can be used to query multiple-stage mass spectra in MS spectral libraries. Additionally the method can be used to calculate structure-spectrum correlations and potentially deduce substructures from spectra of unknown compounds. The approach was tested using two different spectral libraries composed of either human or plant metabolites which currently contain 872 MS(n) spectra acquired from 549 metabolites using Orbitrap FTMS(n). For validation purposes, for 282 of these 549 metabolites, 765 additional replicate MS(n) spectra acquired with the same instrument were used. Both the dereplication and de novo identification functionalities of the comparison approach are discussed. This novel MS(n) spectral processing and comparison approach increases the probability to assign the correct identity to an experimentally obtained fragmentation tree. Ultimately, this tool may pave the way for constructing and populating large MS(n) spectral libraries that can be used for searching and matching experimental MS(n) spectra for annotation and structural elucidation of unknown metabolites detected in untargeted metabolomics studies.

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.

Neutrophil Adhesion and the Release of the Free Amino Acid Hydroxylysine.

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.

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.

Macromolecules of extracellular matrix: determination of selective structures and their functional significance.

In this brief review, I recount events and scientific endeavors in which I have been privileged to participate. The descriptive information includes discovery and characterization of hydroxylysine glycosides from collagen, isolation of dentin sialoprotein (DSP), investigations on dentin phosphoprotein (DPP), and the discovery of a single gene for both DSP and DPP that requires posttranslational proteolytic cleavage of the parent DSPP molecule to generate the two fragments. Finally, I address our unexpected finding of fragments of DMP1 in bone extracts. These fragments are from the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) regions of DMP1. Our studies showed that, similar to DSPP, DMP1 is proteolytically processed by cleavages at X-Asp bonds.

Analysis of collagen and elastin cross-links.

Fibrillar collagens represent the most abundant extracellular matrix proteins in vertebrates providing tissues and organs with form, stability, and connectivity. For such mechanical functions, the formation of covalent intermolecular cross-linking between molecules is essential. This process, the final posttranslational modification during collagen biosynthesis, is initiated by conversion of specific lysine and hydroxylysine residues to the respective aldehydes by the action of lysyl oxidases. This conversion triggers a series of condensation reactions with the juxtaposed lysine-aldehyde, lysine, hydroxylysine, and histidine residues within the same and neighboring molecules resulting in di-, tri-, and tetravalent cross-links. Elastin, another class of extracellular matrix protein, is also stabilized by the lysyl oxidase-mediated mechanism but involving only lysine residues leading to the formation of unique tetravalent cross-links. This chapter presents an overview of fibrillar collagen cross-linking, and the analytical methods for collagen and elastin cross-links we have developed.