Regular ArticleCharacterization of Human and Mouse Cartilage Oligomeric Matrix Protein
Abstract
Cartilage oligomeric matrix protein (COMP) is a 524,000-Da protein that is expressed at high levels in the territorial matrix of chondrocytes. The sequences of rat and bovine COMP indicate that it is a member of the thrombospondin gene family. In this study, we have cloned and sequenced human COMP. Phylogenetic analysis using progressive sequence alignment and two parsimony-based algorithms indicates that the COMP gene and a precursor of the thrombospondin-3 and -4 genes were produced by a gene duplication that occurred 750 million years ago. An interspecific backcross mapping panel has been used to map the murine COMP gene to the central region of mouse chromosome 8. Southern blot analysis of a somatic cell hybrid DNA panel and in situ hybridization to human metaphase chromosomes indicate that the human COMP gene is located on chromosome 19 in band p13.1. These data confirm and extend the known regions of homology between human and mouse chromosomes and establish that COMP, like thrombospondin-1, -2, -3, and -4, is present in the human and mouse genomes.
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DFATs derived from infrapatellar fat pad hold advantage on chondrogenesis and adipogenesis to evade age mediated influence
2023, Journal of Orthopaedic TranslationDedifferentiated fat cells (DFATs) are highly homogeneous and multipotent compared with adipose-derived stromal cells (SCs). Infrapatellar fat pad (IFP)–SCs have advanced chondrogenic potency; however, whether IFP-DFATs could serve as better cell material remains unclear. Here, we aimed to examine the influence of age and body mass index (BMI) on the features of IFPs and IFP-derived cells (IFP–SCs and IFP-DFATs) with exploration of the clinical utilization of IFP-DFATs.
We collected IFPs with isolation of paired IFP-SCs and IFP-DFATs from individuals aged 65 years and older with distinct body weights who underwent total knee replacement for osteoarthritis (OA). Flow cytometry was used to characterize the cellular immunophenotypes. Adipogenesis and chondrogenesis were performed in vitro. Real-time qPCR, western blotting, and Oil Red O or Alcian blue staining were performed to evaluate inflammation, adipogenesis, and chondrogenesis. RNA sequencing and Seahorse analyses were conducted to explore the underlying mechanisms.
We found that IFPs from old or normal-weight individuals with knee OA were pro-inflammatory, and that interleukin-6 (IL-6) signaling was associated with multiple immune-related molecules, whereas IFP-derived cells could escape the inflammatory properties. Aging plays an important role in diminishing the chondrogenic and adipogenic abilities of IFP-SCs; however, this effect was avoided in IFP-DFATs. Generally, IFP-DFATs presented a steady state of chondrogenesis (less influenced by age) and consistently enhanced adipogenesis compared to paired IFP-SCs in different age or BMI groups. RNA sequencing and Seahorse analysis suggested that the downregulation of eukaryotic initiation factor 2 (EIF2) signaling and enhanced mitochondrial function may contribute to the improved cellular biology of IFP-DFATs.
Our data indicate that IFP-DFATs are superior cell material compared to IFP-SCs for cartilage differentiation and adipogenesis, particularly in advanced aging patients with knee OA.
These results provide a novel concept and supportive evidence for the use of IFP-DFATs for cell therapy or tissue engineering in patients with knee OA. Using Ingenuity Pathway Analysis (IPA) of RNA-seq data and Seahorse analysis of mitochondrial metabolic parameters, we highlighted that some molecules, signaling pathways, and mitochondrial functions are likely to be jointly coordinated to determine the enhanced biological function in IFP-DFATs.
Collagen chaperones
2023, Biochemistry of Collagens, Laminins and Elastin: Structure, Function and Biomarkers, Third EditionCollagen biosynthesis requires many interacting components, such as enzymes, proteins, and chaperones. Collagen prolyl hydroxylases are the key enzymes in collagen synthesis that act through the hydroxylation of proline residues in procollagen, which is necessary for the stability of the triple-helical structure. Peptidyl-prolyl cis-trans isomerases are needed to convert all prolines to the trans conformation in a reaction that is also a rate-limiting step in collagen synthesis. The main functions of chaperones are to protect proteins from unfolding/misfolding and prevent the formation of large protein aggregates. This chapter describes the most important chaperones and their roles in collagen fibril formation and disease. Heat-shock protein 47 (HSP47), an intracellular collagen chaperone, plays a critical role in the correct folding of procollagens. The binding of HSP47 molecules to procollagen ensures stabilization while preventing fibril formation and aggregation during transport from the endoplasmic reticulum to the Golgi. Newly discovered interacting proteins of HSP47 may aid in this chaperoning function. Secreted proteome acidic and rich in cysteine (SPARC) functions as an extracellular collagen chaperone by playing a key role in correcting collagen incorporation in tissues. Periostin acts as an extracellular chaperone for proper type I collagen assembly, and the binding of periostin to collagens during fibril formation is needed to stabilize collagen fibrils. Cartilage oligomeric matrix protein (COMP) promotes fibrillogenesis by binding collagen molecules via its homopentameric structure. Diseases associated with collagen chaperones include osteogenesis imperfecta, multiple epiphyseal dysplasia, pseudoachondroplasia, Ehlers–Danlos syndrome, and Bruck syndrome.
The collagen chaperones
2019, Biochemistry of Collagens, Laminins and Elastin: Structure, Function and BiomarkersHeritable Diseases of Connective Tissue
2016, Kelley and Firestein's Textbook of Rheumatology: Volumes 1-2, Tenth EditionCartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: Interaction, regulation and role in chondrogenesis
2014, Matrix BiologyThrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a ‘lattice’ to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions.
Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity
2014, Matrix BiologyThe thrombospondins (TSPs) are a family of matricellular proteins that regulate cellular phenotype through interactions with a myriad of other proteins and proteoglycans. We have identified a novel interaction of the members of the TSP gene family with stromal interaction molecule 1 (STIM1). This association is robust since it is preserved in Triton X-100, can be detected with multiple anti-TSP-1 and anti-STIM1 antibodies, and is detected in a wide range of cell types. We have also found that STIM1 co-immunoprecipitates with TSP-4 and cartilage oligomeric matrix protein (COMP), and that a recombinant version of the N-terminal domain of STIM1 binds to the signature domain of TSP-1 and COMP. The association of the TSPs with STIM1 is observed in both the presence and absence of calcium indicating that the calcium-dependent conformation of the signature domain of TSPs is not required for binding. Thus, this interaction could occur in the ER under conditions of normal or low calcium concentration. Furthermore, we observed that the expression of COMP in HEK 293 cells decreases STIM1-mediated calcium release activated calcium (CRAC) channel currents and increases arachidonic acid calcium (ARC) channel currents. These data indicate that the TSPs regulate STIM1 function and participate in the reciprocal regulation of two channels that mediate calcium entry into the cell.