Glycosaminoglycan Modification of Decorin Depends on MMP14 Activity and Regulates Collagen Assembly.

Proper processing of collagens COL1 and COL6 is required for normal function of adipose tissue and skeletal muscle. Proteoglycan decorin (DCN) regulates collagen fiber formation. The amino-terminus of DCN is modified with an O-linked glycosaminoglycan (GAG), the function of which has remained unclear. Previously, non-glycanated DCN (ngDCN) was identified as a marker of adipose stromal cells. Here, we identify MMP14 as the metalloprotease that cleaves DCN to generate ngDCN. We demonstrate that mice ubiquitously lacking DCN GAG (ngDCN mice) have reduced matrix rigidity, enlarged adipocytes, fragile skin, as well as skeletal muscle hypotrophy, fibrosis, and dysfunction. Our results indicate that DCN deglycanation results in reduced intracellular DCN-collagen binding and increased production of truncated COL6 chains, leading to aberrant procollagen processing and extracellular localization. This study reveals that the GAG of DCN functions to regulate collagen assembly in adipose tissue and skeletal muscle and uncovers a new mechanism of matrix dysfunction in obesity and aging.

Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.

Collagen Stiffness and Architecture Regulate Fibrotic Gene Expression in Engineered Adipose Tissue.

Adipose tissue (AT) has a dynamic extracellular matrix (ECM) surrounding adipocytes that allows for remodeling during metabolic fluctuations. During the progression of obesity, AT has increased ECM deposition, stiffening, and remodeling, resulting in a pro-fibrotic dysfunctional state. Here, the incorporation of ethylene glycol-bis-succinic acid N-hydroxysuccinimide ester (PEGDS) allows for control over 3D collagen hydrogel stiffness and architecture to investigate its influence on adipocyte metabolic and fibrotic function. Upon stiffening and altering ECM architecture, adipocytes did not alter their expression of key adipokines, leptin, and adiponectin. However, they do increase actin cytoskeletal fiber formation, pro-fibrotic gene expression, ECM deposition, and remodeling within a stiffer, 3D collagen hydrogel. For example, COL6A3 gene expression is upregulated approximately twofold, resulting in increased deposition of pericellular collagen VI alpha 3 surrounding adipocytes. Furthermore, inhibition of actin contractility results in a reversal of pro-fibrotic gene expression and ECM deposition, indicating that adipocytes are mediating mechanical cues through actin cytoskeletal networks. This study demonstrates that ECM stiffness and architecture plays a critical regulatory role in adipocyte fibrotic function and contributes to the overall pro-fibrotic dysfunctional state of AT during the progression of obesity and AT fibrosis.

Novel defects in collagen XII and VI expand the mixed myopathy/Ehlers-Danlos syndrome spectrum and lead to variant-specific alterations in the extracellular matrix.

PURPOSE: To date, heterozygous or homozygous COL12A1 variants have been reported in 13 patients presenting with a clinical phenotype overlapping with collagen VI-related myopathies and Ehlers-Danlos syndrome (EDS). The small number of reported patients limits thorough investigation of this newly identified syndrome, currently coined as myopathic EDS. METHODS: DNA from 78 genetically unresolved patients fulfilling the clinical criteria for myopathic EDS was sequenced using a next-generation panel of COL12A1, COL6A1, COL6A2, and COL6A3. RESULTS: Among this cohort, we identified four pathogenic heterozygous in-frame exon skipping (∆) defects in COL12A1, clustering to the thrombospondin N-terminal region and the adjacent collagenous domain (Δ52, Δ53, Δ54, and Δ56 respectively), one heterozygous COL12A1 arginine-to-cysteine substitution of unclear significance (p.(Arg1863Cys)), and compound heterozygous pathogenic COL6A1 variants (c.[98-6G>A];[301C>T]) in one proband. Variant-specific intracellular accumulation of collagen XII chains, extracellular overmodification of the long isoform and near-absence of the short isoform of collagen XII, and extracellular decrease of decorin and tenascin-X were observed for the COL12A1 variants. In contrast, the COL6A1 variants abolished collagen VI and V deposition and increased tenascin-X levels. CONCLUSION: Our data further support the significant clinical overlap between myopathic EDS and collagen VI-related myopathies, and emphasize the variant-specific consequences of collagen XII defects.

Identification of a common epitope in the sequences of COL4A1 and COL6A1 recognized by monoclonal antibody #141.

Identification of a type IV collagen α1 polypeptide in non-triple helical form [NTH α1(IV)], possibly involved in angiogenesis, introduces the further possibility of the existence of non-triple helical forms of other collagen chains. We previously reported that an anti-NTH α1(IV) monoclonal antibody #141 recognizes not only NTH α1(IV) but also a novel non-triple helical collagen polypeptide NTH α1(VI) encoded by COL6A1. In this study, we identified the recognition sequence in order to better understand the properties of antibody #141 and provide clues regarding the biological function of the two non-triple helical molecules. Additionally, we determined the common epitope between COL4A1 and COL6A1 as PXXGXPGLRG, with surface plasmon resonance analyses revealing KD values for the COL4A1 epitope as 5.56±1.81×10-9 M and for the COL6A1 epitope as 7.15±0.44×10-10 M. The specific recognition of NTH α1(IV) and NTH α1(VI) by antibody #141 can be explained by the common epitope sequence. Moreover, epitope localization supports previous finding that NTH α1(IV) and NTH α1(VI) differ in conformation from the α1 chains in triple-helical type IV and type VI collagen. These findings suggest that antibody #141 might be useful for diagnosis of type VI collagen myopathies.

Injectable, redox-polymerized carboxymethylcellulose hydrogels promote nucleus pulposus-like extracellular matrix elaboration by human MSCs in a cell density-dependent manner.

Low back pain is a major cause for disability and is closely linked to intervertebral disc degeneration. Mechanical and biological dysfunction of the nucleus pulposus in the disc has been found to initiate intradiscal degenerative processes. Replacing or enriching the diseased nucleus pulposus with an injectable, stem cell-laden biomaterial that mimics its material properties can provide a minimally invasive strategy for biological and structural repair of the tissue. In this study, injectable, in situ-gelling carboxymethylcellulose hydrogels were developed for nucleus pulposus tissue engineering using encapsulated human marrow-derived mesenchymal stromal cells (hMSCs). With the goal of obtaining robust extracellular matrix deposition and faster construct maturation, two cell-seeding densities, 20 × 106 cells/ml and 40 × 106 cells/ml, were examined. The constructs were fabricated using a redox initiation system to yield covalently crosslinked, cell-seeded hydrogels via radical polymerization. Chondrogenic culture of the hydrogels over 35 days exhibited high cell viability along with deposition of proteoglycan and collagen-rich extracellular matrix, and mechanical and swelling properties similar to native human nucleus pulposus. Further, the matrix production and distribution in the carboxymethylcellulose hydrogels was found to be strongly influenced by hMSC-seeding density, with the lower cell-seeding density yielding a more favorable nucleus pulposus-specific matrix phenotype, while the rate of construct maturation was less dependent on the cell-seeding density. These findings are the first to demonstrate the utility of redox-polymerized carboxymethylcellulose hydrogels as hMSC carriers for potential minimally invasive treatment strategies for nucleus pulposus replacement.

Type VI collagen α1 chain polypeptide in non-triple helical form is an alternative gene product of COL6A1.

Expression of type IV collagen α1 chain in non-triple helical form, NTH α1(IV), is observed in cultured human cells, human placenta and rabbit tissues. Biological functions of NTH α1(IV) are most likely to be distinct from type IV collagen, since their biochemical characteristics are quite different. To explore the biological functions of NTH α1(IV), we prepared some anti-NTH α1(IV) antibodies. In the course of characterization of these antibodies, one antibody, #141, bound to a polypeptide of 140 kDa in size in addition to NTH α1(IV). In this study, we show evidence that the 140 kDa polypeptide is a novel non-triple helical polypeptide of type VI collagen α1 chain encoded by COL6A1, or NTH α1(VI). Expression of NTH α1(VI) is observed in supernatants of several human cancer cell lines, suggesting that the NTH α1(VI) might be involved in tumourigenesis. Reactivity with lectins indicates that sugar chains of NTH α1(VI) are different from those of the α1(VI) chain in triple helical form of type VI collagen, suggesting a synthetic mechanism and a mode of action of NTH α1(VI) is different from type VI collagen.

Collagen VI disorders: Insights on form and function in the extracellular matrix and beyond.

Mutations in the three canonical collagen VI genes, COL6A1, COL6A2 and COL6A3, cause a spectrum of muscle disease from Bethlem myopathy at the mild end to the severe Ullrich congenital muscular dystrophy. Mutations can be either dominant or recessive and the resulting clinical severity is influenced by the way mutations impact the complex collagen VI assembly process. Most mutations are found towards the N-terminus of the triple helical collagenous domain and compromise extracellular microfibril assembly. Outside the triple helix collagen VI is highly polymorphic and discriminating mutations from rare benign changes remains a major diagnostic challenge. Collagen VI deficiency alters extracellular matrix structure and biomechanical properties and leads to increased apoptosis and oxidative stress, decreased autophagy, and impaired muscle regeneration. Therapies that target these downstream consequences have been tested in a collagen VI null mouse and also in small human trials where they show modest clinical efficacy. An important role for collagen VI in obesity, cancer and diabetes is emerging. A major barrier to developing effective therapies is the paucity of information about how collagen VI deficiency in the extracellular matrix signals the final downstream consequences - the receptors involved and the intracellular messengers await further characterization.

A potential role for endogenous proteins as sacrificial sunscreens and antioxidants in human tissues.

Excessive ultraviolet radiation (UVR) exposure of the skin is associated with adverse clinical outcomes. Although both exogenous sunscreens and endogenous tissue components (including melanins and tryptophan-derived compounds) reduce UVR penetration, the role of endogenous proteins in absorbing environmental UV wavelengths is poorly defined. Having previously demonstrated that proteins which are rich in UVR-absorbing amino acid residues are readily degraded by broadband UVB-radiation (containing UVA, UVB and UVC wavelengths) here we hypothesised that UV chromophore (Cys, Trp and Tyr) content can predict the susceptibility of structural proteins in skin and the eye to damage by physiologically relevant doses (up to 15.4 J/cm(2)) of solar UVR (95% UVA, 5% UVB). We show that: i) purified suspensions of UV-chromophore-rich fibronectin dimers, fibrillin microfibrils and ß- and γ-lens crystallins undergo solar simulated radiation (SSR)-induced aggregation and/or decomposition and ii) exposure to identical doses of SSR has minimal effect on the size or ultrastructure of UV chromophore-poor tropoelastin, collagen I, collagen VI microfibrils and α-crystallin. If UV chromophore content is a factor in determining protein stability in vivo, we would expect that the tissue distribution of Cys, Trp and Tyr-rich proteins would correlate with regional UVR exposure. From bioinformatic analysis of 244 key structural proteins we identified several biochemically distinct, yet UV chromophore-rich, protein families. The majority of these putative UV-absorbing proteins (including the late cornified envelope proteins, keratin associated proteins, elastic fibre-associated components and ß- and γ-crystallins) are localised and/or particularly abundant in tissues that are exposed to the highest doses of environmental UVR, specifically the stratum corneum, hair, papillary dermis and lens. We therefore propose that UV chromophore-rich proteins are localised in regions of high UVR exposure as a consequence of an evolutionary pressure to express sacrificial protein sunscreens which reduce UVR penetration and hence mitigate tissue damage.

Aberrant mitochondria in a Bethlem myopathy patient with a homozygous amino acid substitution that destabilizes the collagen VI α2(VI) chain.

Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.

Three-dimensional architecture of collagen type VI in the human trabecular meshwork.

PURPOSE: Type VI collagen is a primary component of the extracellular matrix of many connective tissues. It can form distinct aggregates depending on tissue structure, chemical environment, and physiology. In the current study we examine the ultrastructure and mode of aggregation of type VI collagen molecules in the human trabecular meshwork. METHODS: Trabecular meshwork was dissected from donor human eyes, and three-dimensional transmission electron microscopy of type VI collagen aggregates was performed. RESULTS: Electron-dense collagen structures were detected in the human trabecular meshwork and identified as collagen type VI assemblies based on the three-dimensional spatial arrangement of the type VI collagen molecules, the 105-nm axial periodicity of the assemblies themselves, and their characteristic double bands, which arose from the globular domains of the type VI collagen molecules. Sulfated proteoglycans were also seen to associate with the assemblies either with the globular domain or the inner rod-like segments of the tetramers. CONCLUSIONS: No extended structural regularity in the organization of type VI collagen assemblies within the trabecular meshwork was evident, and the lateral separation of the tetramers forming the assemblies varied, as did the angle formed by the main axes of adjacent tetramers. This is potentially reflective of the specific nature of the trabecular meshwork environment, which facilitates aqueous outflow from the eye, and we speculate that extracellular matrix ions and proteins might prevent a more tight packing of type VI collagen tetramers that form the assemblies.

Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future.

A mouse model for dominant collagen VI disorders: heterozygous deletion of Col6a3 Exon 16.

Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the Col6a3 gene. The resulting heterozygous mouse, Col6a3(+/d16), produced comparable amounts of normal Col6a3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The Col6a3(+/d16) mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The Col6a3(+/d16) mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.

Global analysis reveals the complexity of the human glomerular extracellular matrix.

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.

Design of net-charged abc-type collagen heterotrimers.

Net-negatively-charged heterospecific A:B:C collagen peptide heterotrimers were designed using an automated computational approach. The design algorithm considers both target stability and the energy gap between the target states and misfolded competing states. Structural characterization indicates the net-negative charge balance on the new designs enhances the specificity of the target state at the expense of its stability.

A structure of a collagen VI VWA domain displays N and C termini at opposite sides of the protein.

Von Willebrand factor A (VWA) domains are versatile protein interaction domains with N and C termini in close proximity placing spatial constraints on overall protein structure. The 1.2 Å crystal structures of a collagen VI VWA domain and a disease-causing point mutant show C-terminal extensions that place the N and C termini at opposite ends. This allows a "beads-on-a-string" arrangement of multiple VWA domains as observed for ten N-terminal domains of the collagen VI α3 chain. The extension is linked to the core domain by a salt bridge and two hydrophobic patches. Comparison of the wild-type and a muscular dystrophy-associated mutant structure identifies a potential perturbation of a protein interaction interface and indeed, the secretion of mutant collagen VI tetramers is affected. Homology modeling is used to locate a number of disease-associated mutations and analyze their structural impact, which will allow mechanistic analysis of collagen-VI-associated muscular dystrophy phenotypes.

Position of glycine substitutions in the triple helix of COL6A1, COL6A2, and COL6A3 is correlated with severity and mode of inheritance in collagen VI myopathies.

Glycine substitutions in the conserved Gly-X-Y motif in the triple helical (TH) domain of collagen VI are the most commonly identified mutations in the collagen VI myopathies including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate (INT) phenotypes. We describe clinical and genetic characteristics of 97 individuals with glycine substitutions in the TH domain of COL6A1, COL6A2, or COL6A3 and add a review of 97 published cases, for a total of 194 cases. Clinical findings include severe, INT, and mild phenotypes even from patients with identical mutations. INT phenotypes were most common, accounting for almost half of patients, emphasizing the importance of INT phenotypes to the overall phenotypic spectrum. Glycine substitutions in the TH domain are heavily clustered in a short segment N-terminal to the 17th Gly-X-Y triplet, where they are acting as dominants. The most severe cases are clustered in an even smaller region including Gly-X-Y triplets 10-15, accounting for only 5% of the TH domain. Our findings suggest that clustering of glycine substitutions in the N-terminal region of collagen VI is not based on features of the primary sequence. We hypothesize that this region may represent a functional domain within the triple helix.

Inhibition of SMG-8, a subunit of SMG-1 kinase, ameliorates nonsense-mediated mRNA decay-exacerbated mutant phenotypes without cytotoxicity.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that eliminates aberrant mRNAs containing premature termination codons (PTCs). NMD inhibits the production of aberrant proteins that still retain, at least in part, wild-type function as well as dominant-negative peptides. Therefore, the selective inhibition of NMD has the potential to ameliorate NMD-exacerbated mutant phenotypes. However, we do not have sufficient knowledge of how to effectively suppress NMD with minimum cytotoxic effects. In this study, we aimed to identify NMD-related factors that can be targeted to efficiently inhibit NMD without causing significant cytotoxicity to restore the levels of truncated but partially functional proteins. We evaluated the knockdown of 15 NMD components in Ullrich congenital muscular dystrophy fibroblasts, which have a homozygous frameshift mutation causing a PTC in the collagen type VI α 2 gene. Of the 15 NMD factors tested, knockdown of SMG-8 produced the best effect for restoring defective mRNA and protein levels without affecting cell growth, cell-cycle progression, or endoplasmic reticulum stress. The efficacy of SMG-8 knockdown to improve the mutant phenotype was confirmed using another cell line, from a cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy patient who carries a PTC-containing mutation in HtrA serine peptidase 1. Our results suggest that SMG-8 is an appropriate target for inhibiting NMD to improve NMD-exacerbated mutant phenotypes. NMD inhibition by knockdown of SMG-8 may also be useful to induce synergy in combining the use of read-through drugs for patients with nonsense mutation-associated diseases.