Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence.

Basement membrane collagen IV: Isolation of functional domains.

Collagen IV is a major constituent of basement membranes, specialized form of extracellular matrix that provides a mechanical support for tissues, serves as a polyvalent ligand for cell adhesion receptors and as a scaffold for other proteins, and plays a key role in tissue genesis, differentiation, homeostasis, and remodeling. Collagen IV underlies the pathogenesis of several human disorders including Goodpasture's disease, Alport's syndrome, diabetic nephropathy, angiopathy, and porencephaly. While the isolation of the collagen IV molecules from tissues is an ultimate prerequisite for structural and functional studies, it has been always hampered by the protein insolubility due to extensive intermolecular crosslinking and noncovalent associations with other components of basement membranes. In this chapter, we present methods for the isolation of collagen IV fragments from basement membranes or from extracellular matrix deposited by cultured cells, and the recombinant expression alternative. These methods are useful to address the fundamental questions on the role of collagen IV in tissue genesis under the normal and pathological conditions.

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

BACKGROUND: Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. OBJECTIVES: The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. METHODS: Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. RESULTS: Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. CONCLUSIONS: Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. LEVEL OF EVIDENCE: 5.

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry.

Collagen IV is the main structural protein that provides a scaffold for assembly of basement membrane proteins. Posttranslational modifications such as hydroxylation of proline and lysine and glycosylation of lysine are essential for the functioning of collagen IV triple-helical molecules. These modifications are highly abundant posing a difficult challenge for in-depth characterization of collagen IV using conventional proteomics approaches. Herein, we implemented an integrated pipeline combining high-resolution mass spectrometry with different fragmentation techniques and an optimized bioinformatics workflow to study posttranslational modifications in mouse collagen IV. We achieved 82% sequence coverage for the α1 chain, mapping 39 glycosylated hydroxylysine, 148 4-hydroxyproline, and seven 3-hydroxyproline residues. Further, we employed our pipeline to map the modifications on human collagen IV and achieved 85% sequence coverage for the α1 chain, mapping 35 glycosylated hydroxylysine, 163 4-hydroxyproline, and 14 3-hydroxyproline residues. Although lysine glycosylation heterogeneity was observed in both mouse and human, 21 conserved sites were identified. Likewise, five 3-hydroxyproline residues were conserved between mouse and human, suggesting that these modification sites are important for collagen IV function. Collectively, these are the first comprehensive maps of hydroxylation and glycosylation sites in collagen IV, which lay the foundation for dissecting the key role of these modifications in health and disease.

Identification of a novel COL4A5 mutation in a Chinese family with X-linked Alport syndrome using exome sequencing.

Alport syndrome (AS) is an inherited disorder and clinically characterized by glomerulonephritis and end-stage kidney disease (ESRD). The aim of this study was to identify the gene responsible for glomerulopathy in a 4-generation Chinese pedigree. Exome sequencing was conducted in four patients of the family, and then direct sequencing was performed in other members of the pedigree. A novel missense mutation c.368G>A (p.Gly123Glu) in the collagen type IV alpha-5 gene (COL4A5) was found to be the genetic cause. The p.Gly123Glu mutation occurs prior to Gly-X-Y repeats in the alpha-5 chain of type IV collagen. Neither sensorineural hearing loss nor ocular abnormalities were present in patients of this family. Other clinical features, such as age of onset, age of ESRD, disease severity and complications, varied among patients of this family. Our finding may provide new insights into the cause and diagnosis of AS, and also have implications for genetic counseling.

Developments in purification methods for obtaining and evaluation of collagen derived endogenous angioinhibitors.

Collagen constitutes one of the vital components of the basement membrane scaffolds. Non-collagenous domains (NC1) derived from collagens exhibit potent anti-angiogenic properties, thus attaining significance in regulation of angiogenesis promoted diseases. Individual NC1 domains essential for anti-angiogenic evaluations are generally obtained through purification of individual non-collagenous domains, which have undergone steady developments for enhancing the yields, purpose of biological evaluations and solubility based on the nature of different NC1 domains. This review focuses on the method developments in obtaining biologically active NC1 domains and for specific evaluations in different scenarios.

Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris.

Canstatin-N DNA fragment amplified from human genome was inserted into the MCS of pGAP9K*, an intracellular expression vector of Pichia pastoris, to generate pGAP9K*-can-N which was then transformed into P. pastoris GS115 by electroporation. A transformant was chosen as an engineering strain from the plate containing G418 (700 µg/ml). D-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50 L bioreactor at a 20 L working volume. After 48 h fermentation with continuous feeding of 25% (w/v) D-sorbitol and 0.8% PTM4, the cell grew to A(600)=178 and intracellularly expressed Canstatin-N reached 780 mg/L. Snail enzyme was combined with water to crack P. pastoris and to release intracellular proteins. The purified recombinant Canstatin-N inhibited CAM angiogenesis and induced significant apoptosis of the human umbilical vein endothelial cell (EVC340).

L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

Development of an ELISA for quantification of tumstatin in serum samples and tissue extracts of patients with lung carcinoma.

BACKGROUND: Tumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples. METHODS: Recombinant tumstatin as an immunogen was expressed by E.coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies. RESULTS: The detection limit of the ELISA was 1.4ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7-115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases (P=0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues (P<0.001). CONCLUSIONS: The development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.

The expression of tumstatin is down-regulated in renal carcinoma.

Tumstatin is the 28 kDa NC1 domain of the alpha3 chain of type IV collagen that inhibits pathological angiogenesis and suppresses endothelial cell proliferation and tumor growth. In the present paper, we expressed and purified recombinant human tumstatin protein and then prepared the anti-tumstatin polyclonal antibody. To investigate the expression of tumstatin in renal carcinoma, tumstatin protein was detected by western blotting using the prepared anti-tumstatin antibody and tumstatin mRNA levels were assayed by RT-PCR. The results showed that the expression of tumstatin gene was down-regulated in renal carcinoma tissues and cells. Our study suggests that as a novel endogenous angiogenesis inhibitor, tumstatin gene expression may be a marker for diagnosis, therapy and prognosis of renal carcinoma.

Mitochondria-mediated tumstatin peptide-induced HepG2 cell apoptosis.

The effect of a 19-amino-acid C-terminal peptide of tumstatin (aa 185-203, peptide 19) on human hepatoma cell (HepG2) proliferation was studied, as well as the mechanism by which it induces tumor cell apoptosis. Recombinant peptide 19 was purified by chitin affinity chromatography and identified by Tricine-SDS-PAGE. The DTT was removed with sephadex G-10. MTT colorimetry was used to evaluate the proliferation of tumor cells. Hematoxylin and eosin staining (H&E staining) and AO/EB double staining were used to view morphological changes during apoptosis. Mitochondrial potential was measured via flow cytometer. Western blot analysis was performed to detect the transfer of cytochrome C from mitochondria to the cytoplasm and to monitor the expression levels of caspase-8, caspase-9, Fas, p53, Bcl-2, Bax and Bid in human hepatoma cells. Recombinant peptide 19 effectively suppressed the proliferation of HepG2 cells and induced apoptosis. Each of the two effects had a dose-dependent relationship with recombinant peptide 19. Peptide 19 upregulated the expression of caspase-9, Fas, p53, Bax and Bid, downregulated the expression of Bcl-2 and had little effect on the expression of caspase 8. Peptide 19 decreased the mitochondrial membrane potential and induced the release of cytochrome C from mitochondria to the cytoplasm. In conclusion, peptide 19 induced HepG2 cell apoptosis through the mitochondrial apoptosis pathway.

Expression and In vitro activity of recombinant canstatin in stably transformed bombyx mori cells.

We describe the expression of recombinant canstatin from stably transformed Bombyx mori Bm5 (Bm5) cells. Recombinant canstatin was secreted into a culture medium with a molecular mass of approximately 29 kDa. Densitometric scanning showed that the secreted canstatin accounted for approximately 91% of the total canstatin production. Recombinant canstatin was also purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. The identity of the purified protein was confirmed as human canstatin by nano-LC-MS/MS analysis. Purified recombinant canstatin inhibited human endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant canstatin expressed in stably transformed Bm5 cells was approximately 0.64 mg/ml. A maximum production level of 11 mg/l recombinant canstatin was obtained in a T-flask culture of Bm5 cells after 6 days of incubation.

Expression of soluble, biologically active recombinant human tumstatin in Escherichia coli.

Tumstatin, a 28-kDa C-terminal fragment of collagen IV, is a potent anti-angiogenic protein and inhibitor of tumour growth. Recombinant tumstatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human tumstatin in E. coli by the coding region of tumstatin being linked to the 3'-end of the maltose-binding protein (MBP) gene. The fusion protein was expressed as the soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tumstatin was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 20% of the total soluble protein in E. coli, allowing approximately 8.6 mg of highly purified protein to be obtained per litre of bacterial culture. The purified tumstatin specifically inhibited the proliferation of endothelial cells in a dose-dependent manner. Annexin V-FITC apoptotic assay showed that recombinant tumstatin induced significant increase of apoptotic endothelial cells after 20 h of exposure to 20 microg/ml tumstatin, and when tumstatin was incubated on the chicken embryo, chorioallantoic membrane at doses of 1-15 microg, there was a dramatic decrease in the microvasculature allantoids of chicken embryos neovascular vessel test in vivo demonstrated that tumstatin treatment at doses of 1-15 microg gives rise to dramatically decrease the number of neovascular vessel. Our study provides a feasible and convenient approach to produce soluble and biologically active tumstatin.

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells.

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.

Expression, purification, and bioactivity of human tumstatin from Escherichia coli.

Tumstatin is a M(r) 28,000 C-terminal NC1 fragment of type alpha3 (IV) collagen that inhibits pathological angiogenesis and suppresses proliferation of endothelial cells and growth of tumors. We report here high cytoplasmic expression of recombinant human tumstatin in Escherichia coli and its purification, in vitro refolding, and inhibitory activity analysis. Human tumstatin was expressed in the bacterial cytoplasm as an insoluble N-terminal polyhistidine tagged protein, which accounted for more than 30% of total bacterial protein in BL21 (DE3) cells. After extraction and solubilization in guanidine-HCl, recombinant protein was purified to homogeneity using a simple one-step Ni(2+)-chelate affinity chromatography and then refolded by dialysis against acidic pH buffers with gradually decreasing concentrations of denaturant. The renatured recombinant tumstatin could specifically inhibit endothelial cell proliferation in a dose-dependent manner, and suppress bFGF-induced angiogenesis in chick embryo chorioallantoic membrane and tumor growth in mouse B16 melanoma xenograft models.

The isolation and purification of biologically active recombinant and native autoantigens for the study of autoimmune disease.

The expression of recombinant, biologically active mouse myeloperoxidase (MPD) and the recombinant non-collagenous (NC1) domain of mouse collagen alpha 3 Type IV was achieved for the first time in Sf21 cells (Spodoptera frugiperda ovarian insect cells) using a baculovirus expression system. Following purification, the proteins were identified by reducing and non-reducing SDS-PAGE electrophoresis. Recombinant mouse MPO has a molecular weight of approximately 90 kDa and mouse alpha3(IV)NC1 approximately 32 kDa. In addition, milligram quantities of native mouse myeloperoxidase were purified from 32Dcl3 cells. Both native and recombinant myeloperoxidase were biologically active. This study also demonstrated that the immunization of myeloperoxidase deficient (Mpo-/-) mice with purified recombinant mouse myeloperoxidase induced a significant antibody response to native myeloperoxidase.

[The cloning and expression of angiogenic inhibitor tumstatin(45-132) in E. coli].

AIM: To clone tumstatin(45-132) gene and to express recombinant human tumstatin(45-132) in E. coli BL21. METHODS: Tumstatin gene was cloned by RT-PCR and then tumstatin(45-132) gene amplified from tumstatin gene was cloned into pBV220. The recombinant plasmid pBV220-tumstatin(45-132) was sequenced and transformed into E.coli BL21. E. coli BL21 transformed with the recombinant plasmid pBV220-tumstatin(45-132) was induced at 42 degrees Celsius. After the recombinant tumstatin(45-132) was purified, its bioactivity was detected by endothelia cell proliferation test. RESULTS: 264 bp tumstatin(45-132) fragment was cloned and its sequence was identical with that in GenBank. The tumstatin(45-132) was expressed in E. coli BL21. Expressed product accounted for about 10% of total bacterial proteins and its relative molecular mass (M(r)) was 9,600. The purified protein showed inhibitory effect on proliferation of endothelia cells ECV304 in vitro. CONCLUSION: tumstatin(45-132) gene has been cloned and expressed successfully in E.coli BL21. Expressed tumstatin(45-132) can inhibit the proliferation of endothelial cell ECV304.

Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin.

KTS-disintegrins are a subfamily of short monomeric disintegrins that are potent and selective inhibitors of alpha1beta1 integrin. The amino acid sequence of the new KTS-disintegrin, viperistatin, differs from previously characterized obtustatin in three residues at position 24 (within the integrin binding loop), 38 (hydrophobic core) and 40 (C-terminal region). Noteworthy, viperistatin is about 25-fold more potent than obtustatin inhibiting the binding of this integrin to collagen IV. Synthetic peptides representing the full-length of integrin-binding loops of these disintegrins showed that the Leu24/Arg substitution appears to be partly responsible for the increased inhibitory activity of viperistatin over obtustatin.

Improved production of recombinant tumstatin in stably transformed Trichoplusia ni BTI Tn 5B1-4 cells.

We describe the expression and in vitro activity of recombinant tumstatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells. Recombinant tumstatin was secreted into a culture medium with a molecular weight of 29 kDa. Recombinant tumstatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant tumstatin expressed in stably transformed Tn 5B1-4 cells was approximately 0.76 microg/ml. A maximum production level of 4.0 mg/l recombinant tumstatin was obtained in a T-flask culture of Tn 5B1-4 cells, 6 days after cultivation. We also investigated the individual effects of both dimethyl sulfoxide (DMSO) and sodium butyrate on recombinant tumstatin production in stably transformed Tn 5B1-4 cells. Supplementing cultures with DMSO and sodium butyrate separately increased recombinant tumstatin production in stably transformed Tn 5B1-4 cells by 117 and 32%, respectively.