Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors.
The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)
Almenoff
J S
JS
Howard Hughes Medical Institute, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical Center, California 94305.
Williams
S I
SI
Scheving
L A
LA
Judd
A K
AK
Schoolnik
G K
GK
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000378:metabolism*; D001710:Biotin; D002277:Carcinoma; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006046:Gold; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D007082:Ileum; D007163:Immunosorbent Techniques; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D008024:Ligands; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D051381:Rats; D011956:Receptors, Cell Surface; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D015398:Signal Transduction; D019809:Streptavidin; D014162:Transfection; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008024:Ligands; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D001710:Biotin; D006046:Gold; D019809:Streptavidin; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled