Differential processing of guanylyl cyclase C along villus-crypt axis of rat small intestine

Many strains of enterotoxigenic Escherichia coli produce a heat-stable peptide enterotoxin (STa) that binds to the intestinal receptor guanylyl cyclase C (GC-C). STa receptors are structurally heterogeneous, but the molecular events causing this heterogeneity remain obscure. We examined the influence of cell position along the villus-crypt axis on STa receptor heterogeneity by fractionating EDTA-dissociated cells that detached in a villus-to-crypt direction. STa affinity labeling experiments revealed that the initially released villus “tip” fraction had four major STa binding proteins (STBPs), with relative molecular weight (M(r)) of 150,000, 135,000, 125,000, and 95,000, that did not react with a GC-C carboxy-terminal antibody. Yet succeeding villus cell fractions had major immunoreactive STBPs with M(r) of 275,000 and 250,000. Limited proteolysis of these larger GC-C isoforms produced 1) smaller STBPs that had M(r) similar to those in the initial villus fraction, 2) a 65,000 M(r) protein GC-C isoform that did not bind STa, and 3) elevated basal and STa-induced cyclase activity. Our data show that STBP structural heterogeneity in the intact intestine arises largely from multisite proteolytic processing of GC-C.

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In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies

Molecules ◽

10.3390/molecules26144147 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4147

Author(s):

Neha Gupta ◽

Saurav Kumar Choudhary ◽

Neeta Bhagat ◽

Muthusamy Karthikeyan ◽

Archana Chaturvedi

Keyword(s):

Molecular Docking ◽

Amino Acid ◽

Guanylyl Cyclase ◽

Extracellular Domain ◽

Enterotoxigenic Escherichia Coli ◽

Watery Diarrhea ◽

Guanylyl Cyclase C ◽

Amino Acid Residues ◽

Lead Compounds

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein–protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

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