Design, Preparation, and Bioactivity Study of New Fusion Protein HB-NC4 in the Treatment of Osteoarthritis

Osteoarthritis (OA) is now becoming the main disease that affects public health. There is no specific medicine used for OA in clinical application until now. Recently, several studies demonstrated that OA is closely related to the complement system, and some complement regulators such as N-terminal non-collagenous domain 4 (NC4) aimed at alleviating OA have shown a promising therapeutic effect. However, targeting ability is the main limitation for NC4. In this study, a fusion protein named heparin-binding domain-N-terminal non-collagenous domain 4 (HB-NC4) was proposed to solve this problem, which could provide a better way for OA treatment. First, HB-NC4 plasmid was constructed using ClonExpress II one-step ligation kit method. And Escherichia coli BL21 was utilized to express the fusion protein, Ni2+-sepharose, and a desalting gravity column were introduced to purify HB-NC4. The results showed that 0.84 mg HB-NC4 could be obtained from a 1 L culture medium with a purity higher than 92.6%. Then, the hemolytic assay was introduced to validate the anti-complement activity of HB-NC4; these results demonstrated that both HB-NC4 and NC4 had a similar anti-complement activity, which indicated that heparin-binding (HB) did not affect the NC4 structure. Targeting ability was investigated in vivo. HB-NC4 showed a higher affinity to cartilage tissue than NC4, which could prolong the retention time in cartilage. Finally, the destabilization of the medial meniscus (DMM) model was applied to investigate HB-NC4 pharmacodynamics in vivo. The results indicated that HB-NC4 significantly slowed cartilage degradation during the OA process. In summary, compared with NC4, HB-NC4 had better-targeting ability which could improve its therapeutic effect and prolonged its action time. It could be used as a new complement regulator for the treatment of OA in the future.

Collagen Binding Proteins of Gram-Positive Pathogens

Collagens are the primary structural components of mammalian extracellular matrices. In addition, collagens regulate tissue development, regeneration and host defense through interaction with specific cellular receptors. Their unique triple helix structure, which requires a glycine residue every third amino acid, is the defining structural feature of collagens. There are 28 genetically distinct collagens in humans. In addition, several other unrelated human proteins contain a collagen domain. Gram-positive bacteria of the genera Staphylococcus, Streptococcus, Enterococcus, and Bacillus express cell surface proteins that bind to collagen. These proteins of Gram-positive pathogens are modular proteins that can be classified into different structural families. This review will focus on the different structural families of collagen binding proteins of Gram-positive pathogen. We will describe how these proteins interact with the triple helix in collagens and other host proteins containing a collagenous domain and discuss how these interactions can contribute to the pathogenic processes.

Sequence-dependent mechanics of collagen reflect its structural and functional organization

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagens. In particular, network-forming collagen IV, an integral component of basement membranes, has been far less studied than fibril-forming collagens. A key feature differentiating collagen IV is the presence of interruptions in the triple-helix-defining (Gly-X-Y) sequence along its collagenous domain. Here, we used atomic force microscopy (AFM) to determine the impact of these interruptions on the flexibility of collagen. Our extracted flexibility profile reveals that collagen IV possesses highly heterogeneous mechanics, ranging from semi-flexible regions as found for fibril-forming collagens to a lengthy region of high flexibility towards its N terminus. A simple model in which flexibility is dictated only by the presence of interruptions fit the extracted profile reasonably well, providing insight into the alignment of chains and supporting the role of interruptions in instilling flexibility. However, limitations of this model were illuminated by our determination of variable flexibility along continuously triple-helical collagen III, which we found to possess a high-flexibility region around its matrix-metalloprotease (MMP) binding site. Surprisingly, proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are not used to control its compaction during secretion. Although extracellular chloride ions play a role in triggering collagen IV network formation, they do not appear to modulate the structure of its collagenous domain.

RETRACTED: Production and Characterization of Recombinant Rat Non-collagen Domain of α3 Chain of Type IV Collagen α3 (IV) NC1 Antigen

Glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney which causes kidney diseases. Glomerulonephritis disease deposits the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain, and a C- terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the experimental autoimmuno glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV)NC1 antigen has nine amino acid span that is consistent with antibody or T cell epitope which is induced in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of Anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant which is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column. In addition, it is characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques.