A strategy for enrichment of claudins based on their affinity to Clostridium perfringens enterotoxin.
BACKGROUND
Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes.


RESULTS
Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE(116-319) fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material.


CONCLUSIONS
CPE(116-319) provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.
Lohrberg
Dörte
D
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany. dlohrberg@web.de
Krause
Eberhard
E
Schümann
Michael
M
Piontek
Jörg
J
Winkler
Lars
L
Blasig
Ingolf E
IE
Haseloff
Reiner F
RF
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D003016:Clostridium perfringens / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D019108:Tight Junctions / Q000378:metabolism*
D004768:Enterotoxins; D008565:Membrane Proteins; C035598:enterotoxin, Clostridium