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Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

ABSTRACT: The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.

SUBMITTER: Xiao Y

PROVIDER: S-EPMC1218333 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Project description:Disulphide bonds are stabilizing crosslinks in proteins and serve to enhance their thermal stability. In proteins that are small and rich in disulphide bonds, they could be the major determining factor for the choice of conformational state since their constraints on appropriate backbone conformation can be substantial. Such crosslinks and their positional conservation could itself enable protein family and functional association. Despite the importance of the field, there is no comprehensive database on disulphide crosslinks that is available to the public. Herein we provide information on disulphides in DSDBASE2.0, an updated and significantly expanded database that is freely available, fully annotated and manually curated database on native and modelled disulphides. The web interface also provides several useful computational tools that have been specifically developed for proteins containing disulphide crosslinks. The modelling of disulphide crosslinks is performed using stereochemical criteria, coded within our Modelling of Disulphides in Proteins (MODIP) algorithm. The inclusion of modelled disulphides potentially enhances the loop database substantially, thereby permitting the recognition of compatible polypeptide segments that could serve as templates for immediate modelling. The DSDBASE2.0 database has been updated to include 153,944 PDB entries, 216,096 native and 20,153,850 modelled disulphide bond segments from PDB January 2021 release. The current database also provides a resource to user-friendly search for multiple disulphide bond containing loops, along with annotation of their function using GO and subcellular localization of the query. Furthermore, it is possible to obtain the three-dimensional models of disulphide-rich small proteins using an independent algorithm, RANMOD, that generates and examines random, but allowed backbone conformations of the polypeptide. DSDBASE2.0 still remains the largest open-access repository that organizes all disulphide bonds of proteins on a single platform. The database can be accessed from http://caps.ncbs.res.in/dsdbase2.

Project description:OBJECTIVE:To assess the association between use of sodium-glucose co-transporter 2 (SGLT2) inhibitors and risk of serious renal events in data from routine clinical practice. DESIGN:Cohort study using an active comparator, new user design and nationwide register data. SETTING:Sweden, Denmark, and Norway, 2013-18. PARTICIPANTS:Cohort of 29?887 new users of SGLT2 inhibitors (follow-up time: dapagliflozin 66.1%; empagliflozin 32.6%; canagliflozin 1.3%) and 29?887 new users of an active comparator, dipeptidyl peptidase-4 inhibitors, matched 1:1 on the basis of a propensity score with 57 variables. Mean follow-up time was 1.7 (SD 1.0) years. EXPOSURES:SGLT2 inhibitors versus dipeptidyl peptidase-4 inhibitors, defined by filled prescriptions and analysed according to intention to treat. MAIN OUTCOME MEASURES:The main outcome was serious renal events, a composite including renal replacement therapy, death from renal causes, and hospital admission for renal events. Secondary outcomes were the individual components of the main outcome. RESULTS:The mean age of the study population was 61.3 (SD 10.5) years; 11?108 (19%) had cardiovascular disease, and 1974 (3%) had chronic kidney disease. Use of SGLT2 inhibitors, compared with dipeptidyl peptidase-4 inhibitors, was associated with a reduced risk of serious renal events (2.6 events per 1000 person years versus 6.2 events per 1000 person years; hazard ratio 0.42 (95% confidence interval 0.34 to 0.53); absolute difference -3.6 (-4.4 to -2.8) events per 1000 person years). In secondary outcome analyses, the hazard ratio for use of SGLT2 inhibitors versus dipeptidyl peptidase-4 inhibitors was 0.32 (0.22 to 0.47) for renal replacement therapy, 0.41 (0.32 to 0.52) for hospital admission for renal events, and 0.77 (0.26 to 2.23) for death from renal causes. In sensitivity analyses in each of the Swedish and Danish parts of the cohort, the model was further adjusted for glycated haemoglobin and estimated glomerular filtration rate (Sweden and Denmark) and for blood pressure, body mass index, and smoking (Sweden only); in these analyses, the hazard ratio moved from 0.41 (0.26 to 0.66) to 0.50 (0.31 to 0.81) in Sweden and from 0.42 (0.32 to 0.56) to 0.55 (0.41 to 0.74) in Denmark. CONCLUSIONS:In this analysis using nationwide data from three countries, use of SGLT2 inhibitors, compared with dipeptidyl peptidase-4 inhibitors, was associated with a significantly reduced risk of serious renal events.

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Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

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