Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E₂, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE₂ had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of α1 and α2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the α1 chain, pepsin cleaved between Asp¹⁰³⁵ and Phe¹⁰³⁶, and actinidain between Gly¹⁰³² and Gly¹⁰³³. Thus, the actinidain-hydrolyzed α1 chain is shorter at the C terminus by three residues, Gly¹⁰³³, Phe¹⁰³⁴, and Asp¹⁰³⁵. In the α2 chain, both proteases cleaved between Glu¹⁰³⁰ and Val¹⁰³¹. We demonstrated that a synthetic nonapeptide mimicking the α1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the α1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.     

Breaking the light and heavy chain linkage of human immunoglobulin G1 (IgG1) by radical reactions

We report that the production of hydrogen peroxide by radical chain reductions of molecular oxygen into water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The production of the hydrogen peroxide can be accelerated by superoxide dismutase or redox active metal ions or inhibited by free radical scavengers. The hydrogen peroxide production rate correlates well with the hinge cleavage. In addition to radical reaction mechanisms described previously, new degradation pathways and products were observed. These products were determined to be generated via radical reactions initiated by electron transfer and addition to the interchain disulfide bond between Cys(215) of the light chain and Cys(225) of the heavy chain. Decomposition of the resulting disulfide bond radical anion breaks the C-S bond at the side chain of Cys, converting it into dehydroalanine and generating a sulfur radical adduct at its counterpart. The hydrolysis of the unsaturated dehydropeptides removes Cys and yields an amide at the C terminus of the new fragment. Meanwhile, the competition between the carbonyl (-C(α)ONH-) and the side chain of Cys allows an electron transfer to the α carbon, forming a new intermediate radical species (-(·)C(α)(O(-))NH-) at Cys(225). Dissociative deamidation occurs along the N-C(α) bond, resulting in backbone cleavage. Given that hydrogen peroxide is a commonly observed product of thermal stress and plays a role in mediating the unique degradation of an IgG1, strategies for improving stability of human antibody therapeutics are discussed.     

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme

The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5′ splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.     

Adenovirus Composition,Proteolysis, and Disassembly Studied by In-depth Qualitative and Quantitative Proteomics

Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.     

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid

In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1-42), and N-terminally truncated Aβ(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Interaction with Polyglutamine Aggregates Reveals a Q/N-rich Domain in TDP-43

The identification of pathologic TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, followed by the discovery of dominantly inherited point mutations in TDP-43 in familial ALS, have been critical insights into the mechanism of these untreatable neurodegenerative diseases. However, the biochemical basis of TDP-43 aggregation and the mechanism of how mutations in TDP-43 lead to disease remain enigmatic. In efforts to understand how TDP-43 alters its cellular localization in response to proteotoxic stress, we found that TDP-43 is sequestered into polyglutamine aggregates. Furthermore, we found that binding to polyglutamine aggregates requires a previously uncharacterized glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43. Sequestration into polyglutamine aggregates causes TDP-43 to be cleared from the nucleus and become detergent-insoluble. Finally, we observed that sequestration into polyglutamine aggregates led to loss of TDP-43-mediated splicing in the nucleus and that polyglutamine toxicity could be partially rescued by increasing expression of TDP-43. These data indicate pathologic sequestration into polyglutamine aggregates, and loss of nuclear TDP-43 function may play an unexpected role in polyglutamine disease pathogenesis. Furthermore, as Q/N domains have a strong tendency to self-aggregate and in some cases can function as prions, the identification of a Q/N domain in TDP-43 has important implications for the mechanism of pathologic aggregation of TDP-43 in ALS and other neurodegenerative diseases.     

Osmotic stress inhibits proteasome by p38 MAPK-dependent phosphorylation

Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2.     

Structure-Function Analysis of CCL28 in the Development of Post-viral Asthma

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded β-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.     

Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ～10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.     

Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.     

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.     

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.     

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.     

Sequence Analysis and Domain Motifs in the Porcine Skin Decorin Glycosaminoglycan Chain

Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences.     

The Activity of Hyaluronan Synthase 2 Is Regulated by Dimerization and Ubiquitination

Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag- and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.     

Maturation of Collagen Ketoimine Cross-links by an Alternative Mechanism to Pyridinoline Formation in Cartilage

The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH₄. On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.