Tamm Horsfall protein binds to a single class of carbohydrate specific receptors on human neutrophils.
Several studies have implicated urinary Tamm Horsfall protein (THP) in the aetiology of tubulointerstitial inflammation. Previous research has demonstrated that particulate THP will initiate inflammatory activation of human polymorphonuclear leukocytes (PMN) through a trypsin sensitive mechanism. The present study describes the binding of 125I-monomeric THP to human PMN at 4 degrees C and demonstrates that radiolabeled THP binds to PMN in a saturable, reversible and time dependent manner. The addition of 400 to 600 ng 125I-THP/2 x 10(6) PMN was sufficient to achieve saturable binding. Scatchard analysis of binding data yielded linear plots suggesting a single class of receptors with a mean density of 26218/cell and a dissociation constant (KD) of 4.2 x 10(-9) M. Binding reached steady state by 15 minutes and could be rapidly displaced by the addition of an excess of unlabeled THP. The KD calculated from experimentally derived kinetic rate constants (k1 and k2) was of a similar order of magnitude (0.9 x 10(-9) M) to that generated from Scatchard plots. In addition 125I-THP bound specifically to PMN plasma membrane immobilized on nitrocellulose filters, a process which could be inhibited by unlabeled monomeric THP. Chemical modification of unlabeled THP abolished its capacity to inhibit binding. Specific inhibition studies showed that N-acetylneuraminic (sialic) acid dose dependently displaced the binding of 125I-THP to immobilized PMN membranes at concentrations up to 100 mM. These results indicate that the reported activation of human PMN by THP is mediated through a single class of sialic acid-specific cell surface receptors.
Institute of Nephrology, University of Wales College of Medicine, Cardiff Royal Infirmary, United Kingdom.
D B
DB
Thomas
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M
Davies
J R
JR
Peters
J D
JD
Williams
D050260:Carbohydrate Metabolism; D002462:Cell Membrane / Q000378:metabolism; D001330:Electronic Data Processing; D006801:Humans; D007700:Kinetics; D009005:Monosaccharides / Q000494:pharmacology; D009088:Mucoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000145:classification / Q000378:metabolism*; D013997:Time Factors; D058949:Uromodulin
D009005:Monosaccharides; D009088:Mucoproteins; D011956:Receptors, Cell Surface; C548612:UMOD protein, human; D058949:Uromodulin