Novel Insights into the Catalytic Mechanism of Collagenolysis by Zn(II)-Dependent Matrix Metalloproteinase-1.

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.

Identification of binding partners that facilitate membrane-type 5 matrix metalloproteinase (MT5-MMP) processing of amyloid precursor protein.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular deposits of amyloid beta (Aß) peptide. In addition to Aß as the core component of the amyloid plaque, the amyloid precursor protein (APP) processing fragment Aß was also found accumulated around the plaque. The APPη pathway, mainly mediated by membrane-type 5 matrix metalloproteinase (MT5-MMP), represents an important factor in AD pathogenesis. The proamyloidogenic features of MT5-MMP could result from interactions with APP when trafficking between organelles, so determination of the location within the cell of APPη cleavage and interacting proteins of MT5-MMP affecting this process will be of priority in understanding the role of MT5-MMP in AD. In the present study, MT5-MMP was found to be located in the nucleus, cytosol, and cytosolic subcellular granules of CHO cells that stably expressed wild-type human APP751. MT5-MMP fusion proteins were constructed that could localize enzyme production in the Golgi apparatus, endosome, ER, mitochondria, or plasma membrane. The fusion proteins significantly increased sAPPη when directed to the endosome, Golgi apparatus, plasma membrane, or mitochondria. Since the C-terminal region of MT5-MMP is responsible for its intracellular location and trafficking, this domain was used as the bait in a yeast two-hybrid screen to identify MT5-MMP protein partners in a human brain cDNA library. Identified binding partners included N4BP2L1, TMX3, EIG121, bridging Integrator 1 (BIN1), RUFY4, HTRA1, and TMEM199. The binding of N4BP2L1, EIG121, BIN1, or TMX3 to MT5-MMP resulted in the most significant increase in sAPPη production. Thus, the action of MT5-MMP on APP occurs in multiple locations within the cell and is facilitated by site-specific binding partners.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

The front cover artwork is provided by Dr. Karabencheva-Christova's group at Michigan Technological University. The images show the initially formed and the catalytically productive conformations of MMP-1 complex with the Triple Helical Peptide (THP), the free energy profile connecting them as well as the coordination geometry of the catalytic zinc (II). The background shows the collagen macromolecule. Read the full text of the Research Article at 10.1002/cphc.202200649.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1•THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's α-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

Skeletal muscle proteome expression differentiates severity of cancer cachexia in mice and identifies loss of fragile X mental retardation syndrome-related protein 1.

Tandem mass tag (TMT)-based quantitative proteomics was used to examine protein expression in skeletal muscle from mice with moderate and severe cancer cachexia to study mechanisms underlying varied cachexia severity. Weight loss of 10% (moderate) and 20% (severe) was induced by injection of colon-26 cancer cells in 10-week old Balb/c mice. In moderate cachexia, enriched pathways reflected fibrin formation, integrin/mitogen-activated protein kinase (MAPK) signaling, and innate immune system, suggesting an acute phase response and fibrosis. These pathways remained enriched in severe cachexia; however, energy-yielding pathways housed in mitochondria were prominent additions to the severe state. These enrichments suggest distinct muscle proteome expression patterns that differentiate cachexia severity. When analyzed with two other mouse models, eight differentially expressed targets were shared including serine protease inhibitor A3N (Serpina3n), synaptophysin-like protein 2 (Sypl2), Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial (Idh3a), peroxisomal acyl-coenzyme A oxidase 1 (Acox1), collagen alpha-1(VI) chain (Col6a1), myozenin 3 (Myoz3), UDP-glucose pyrophosphorylase (Ugp2), and solute carrier family 41 member 3 (Slc41a3). Acox1 and Idh3a control lipid oxidation and NADH generation in the TCA cycle, respectively, and Col6a1 comprises part of type VI collagen with reported profibrotic functions, suggesting influential roles in cachexia. A potential target was identified in fragile X mental retardation syndrome-related protein 1 (FXR1), an RNA-binding protein not previously implicated in cancer cachexia. FXR1 decreased in cachexia and related linearly with weight change and myofiber size. These findings suggest distinct mechanisms associated with cachexia severity and potential biomarkers and therapeutic targets.

Development of a putative Zn2+-chelating but highly selective MMP-13 inhibitor.

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.

A synergy between the catalytic and structural Zn(II) ions and the enzyme and substrate dynamics underlies the structure-function relationships of matrix metalloproteinase collagenolysis.

Matrix metalloproteinases (MMPs) are Zn(II) dependent endopeptidases involved in the degradation of collagen. Unbalanced collagen breakdown results in numerous pathological conditions, including cardiovascular and neurodegenerative diseases and tumor growth and invasion. Matrix metalloproteinase-1 (MMP-1) is a member of the MMPs family. The enzyme contains catalytic and structural Zn(II) ions. Despite many studies on the enzyme, there is little known about the synergy between the two Zn(II) metal ions and the enzyme and substrate dynamics in MMP-1 structure-function relationships. We performed a computational study of the MMP-1•triple-helical peptide (THP) enzyme•substrate complex to provide this missing insight. Our results revealed Zn(II) ions' importance in modulating the long-range correlated motions in the MMP-1•THP complex. Overall, our results reveal the importance of the catalytic Zn(II) and the role of the structural Zn(II) ion in preserving the integrity of the enzyme active site and the overall enzyme-substrate complex synergy with the dynamics of the enzyme and the substrate. Notably, both Zn(II) sites participate in diverse networks of long-range correlated motions that involve the CAT and HPX domains and the THP substrate, thus exercising a complex role in the stability and functionality of the MMP-1•THP complex. Both the Zn(II) ions have a distinct impact on the structural stability and dynamics of the MMP-1•THP complex. The study shifts the paradigm from the "local role" of the Zn(II) ions with knowledge about their essential role in the long-range dynamics and stability of the overall enzyme•substrate (ES) complex.

Effects of the Nature of the Metal Ion, Protein and Substrate on the Catalytic Center in Matrix Metalloproteinase-1: Insights from Multilevel MD, QM/MM and QM Studies.

Matrix metalloproteinase-1 (MMP-1) is a Zn(II) dependent endopeptidase involved in the degradation of collagen, the most abundant structural protein in the extracellular matrix of connective tissues and the human body. Herein we performed a multilevel computational analysis including molecular dynamics (MD), combined quantum mechanics/molecular mechanics (QM/MM), and quantum mechanics (QM) calculations to characterize the structure and geometry of the catalytic Zn(II) within the MMP-1 protein environment in comparison to crystallographic and spectroscopic data. The substrate's removal fine-tuned impact on the conformational dynamics and geometry of the catalytic Zn(II) center was also explored. Finally, the study examined the effect of substituting catalytic Zn(II) by Co(II) on the overall structure and dynamics of the MMP-1 THP complex and specifically on the geometry of the catalytic metal center. Overall our QM/MM and QM studies were in good agreement with the MM description of the Zn(II) centers in the MD simulations.

Hepatic proteome analysis reveals altered mitochondrial metabolism and suppressed acyl-CoA synthetase-1 in colon-26 tumor-induced cachexia.

Cachexia is a life-threatening complication of cancer traditionally characterized by weight loss and muscle dysfunction. Cachexia, however, is a systemic disease that also involves remodeling of nonmuscle organs. The liver exerts major control over systemic metabolism, yet its role in cancer cachexia is not well understood. To advance the understanding of how the liver contributes to cancer cachexia, we used quantitative proteomics and bioinformatics to identify hepatic pathways and cellular processes dysregulated in mice with moderate and severe colon-26 tumor-induced cachexia; ~300 differentially expressed proteins identified during the induction of moderate cachexia were also differentially regulated in the transition to severe cachexia. KEGG pathway enrichment revealed representation by oxidative phosphorylation, indicating altered hepatic mitochondrial function as a common feature across cachexia severity. Glycogen catabolism was also observed in cachexic livers along with decreased pyruvate dehydrogenase protein X component (Pdhx), increased lactate dehydrogenase A chain (Ldha), and increased lactate transporter Mct1. Together this suggests altered lactate metabolism and transport in cachexic livers, which may contribute to energetically inefficient interorgan lactate cycling. Acyl-CoA synthetase-1 (ACSL1), known for activating long-chain fatty acids, was decreased in moderate and severe cachexia based on LC-MS/MS analysis and immunoblotting. ACSL1 showed strong linear relationships with percent body weight change and muscle fiber size (R2 = 0.73-0.76, P < 0.01). Mitochondrial coupling efficiency, which is compromised in cachexic livers to potentially increase energy expenditure and weight loss, also showed a linear relationship with ACSL1. Findings suggest altered mitochondrial and substrate metabolism of the liver in cancer cachexia, and possible hepatic targets for intervention.

TF-containing MUC1 glycopeptides fail to entice Galectin-1 recognition of tumor-associated Thomsen-Freidenreich (TF) antigen (CD176) in solution.

Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1's tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.

Dissecting MMP P10' and P11' subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that remodel the extracellular matrix environment and mitigate outside-in signaling. Loss of regulation of MMP activity plays a role in numerous pathological states. In particular, aberrant collagenolysis affects tumor invasion and metastasis, osteoarthritis, and cardiovascular and neurodegenerative diseases. To evaluate the collagen sequence preferences of MMPs, a positional scanning synthetic combinatorial library was synthesized herein and was used to investigate the P10' and P11' substrate subsites. The scaffold for the library was a triple-helical peptide mimic of the MMP cleavage site in types I-III collagen. A FRET-based enzyme activity assay was used to evaluate the sequence preferences of eight MMPs. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. On the basis of the screening results, several individual peptides were designed and evaluated. A triple-helical substrate incorporating Asp-Lys in the P10'-P11' subsites offered selectivity between MMP-14 and MMP-15, whereas Asp-Lys or Trp-Lys in these subsites discriminated between MMP-2 and MMP-9. Future screening of additional subsite positions will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery. Furthermore, selective substrates could serve as the basis for the design of specific triple-helical peptide inhibitors targeting only those MMPs that play a detrimental role in a disease of interest.

A Novel Probe for Spliceosomal Proteins that Induces Autophagy and Death of Melanoma Cells Reveals New Targets for Melanoma Drug Discovery.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.

Matrix Metalloproteinase Triple-Helical Peptide Inhibitors: Potential Cross-Reactivity with Caspase-11.

Triple-helical peptide inhibitors (THPIs) of matrix metalloproteinases (MMPs) have recently been demonstrated to be effective in a variety of animal models of disease, coincidental with knockout studies. However, passenger mutations have been described in MMP knockout mice that impact the activity of other proteins, including caspase-11. Thus, it is possible that the results observed with THPIs may be based on inhibition of caspase-11, not MMPs. The present study evaluated whether THPIs were cross-reactive with caspase-11. Two different THPIs were tested, one that is known to inhibit MMP-1 and MMP-8 (GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI) and one that is selective for MMP-2 and MMP-9 (α1(V)GlyΨ{PO2H-CH2}Val [mep14,32,Flp15,33] THPI). No inhibition of caspase-11 was observed with GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI, even at an inhibitor concentration of 5 µM, while 5 µM α1(V)GlyΨ{PO2H-CH2}Val [mep14,32,Flp15,33] THPI exhibited 40% inhibition of caspase-11. Further testing of GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI revealed nM inhibition of MMP-2, MMP-9, and MMP-13. Thus, the effectiveness of GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI observed in a sepsis animal model may not be due to caspase-11 inhibition, but may be due to broader MMP inhibition than previously thought.

Matrix metalloproteinase collagenolysis in health and disease.

The proteolytic processing of collagen (collagenolysis) is critical in development and homeostasis, but also contributes to numerous pathologies. Mammalian interstitial collagenolytic enzymes include members of the matrix metalloproteinase (MMP) family and cathepsin K. While MMPs have long been recognized for their ability to catalyze the hydrolysis of collagen, the roles of individual MMPs in physiological and pathological collagenolysis are less defined. The use of knockout and mutant animal models, which reflect human diseases, has revealed distinct collagenolytic roles for MT1-MMP and MMP-13. A better understanding of temporal and spatial collagen processing, along with the knowledge of the specific MMP involved, will ultimately lead to more effective treatments for cancer, arthritis, cardiovascular conditions, and infectious diseases. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Development of matrix metalloproteinase-13 inhibitors - A structure-activity/structure-property relationship study.

A structure-activity/structure-property relationship study based on the physicochemical as well as in vitro pharmacokinetic properties of a first generation matrix metalloproteinase (MMP)-13 inhibitor (2) was undertaken. After systematic variation of inhibitor 2, compound 31 was identified which exhibited microsomal half-life higher than 20 min, kinetic solubility higher than 20 µM, and a permeability coefficient greater than 20 × 10-6 cm/s. Compound 31 also showed excellent in vivo PK properties after IV dosing (Cmax = 56.8 µM, T1/2 (plasma) = 3.0 h, Cl = 0.23 mL/min/kg) and thus is a suitable candidate for in vivo efficacy studies in an OA animal model.

Tricine as a convenient scaffold for the synthesis of C-terminally branched collagen-model peptides.

A novel and convenient method for the synthesis of C-terminally branched collagen-model peptides has been achieved using tricine (N-[tris(hydroxymethyl)methyl]glycine) as a branching scaffold and 1,2-diaminoethane or 1,4-diaminobutane as a linker. The peptide sequence was incorporated directly onto the linker and scaffold during solid-phase synthesis without additional manipulations. The resulting branched triple-helical peptides exhibited comparable thermal stabilities to the parent, unbranched sequence, and served as substrates for matrix metalloproteinase-1 (MMP-1). The tricine-based branch reported herein represents the simplest synthetic scaffold for the convenient synthesis of covalently linked homomeric collagen-model triple-helical peptides.

Clinical Implications of Compounds Designed to Inhibit ECM-Modifying Metalloproteinases.

Remodeling of the extracellular matrix (ECM) is crucial in development and homeostasis, but also has a significant role in disease progression. Two metalloproteinase families, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs), participate in the remodeling of the ECM, either directly or through the liberation of growth factors and cell surface receptors. The correlation of MMP and ADAM activity to a variety of diseases has instigated numerous drug development programs. However, broad-based and Zn2+ -chelating MMP and ADAM inhibitors have fared poorly in the clinic. Selective MMP and ADAM inhibitors have been described recently based on (a) antibodies or antibody fragments or (b) small molecules designed to take advantage of protease secondary binding sites (exosites) or allosteric sites. Clinical trials have been undertaken with several of these inhibitors, while others are in advanced pre-clinical stages.

Path to Collagenolysis: COLLAGEN V TRIPLE-HELIX MODEL BOUND PRODUCTIVELY AND IN ENCOUNTERS BY MATRIX METALLOPROTEINASE-12.

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å(2)of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling.

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

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