Tamm-Horsfall glycoprotein enhances PMN phagocytosis by binding to cell surface-expressed lactoferrin and cathepsin G that activates MAP kinase pathway.

Syue-Cian Siao,Chang-Youh Tsai,Ko-Jen Li,Cheng-Han Wu,Chia-Li Yu,Ming-Chi Lu,Song-Chou Hsieh

doi:10.3390/molecules16032119

Abstract

The molecular basis of polymorphonuclear neutrophil (PMN) phagocytosis-enhancing activity (PEA) by human purified urinary Tamm-Horsfall glyco- protein (THP) has not been elucidated. In this study, we found human THP bound to lactoferrin (LF) and cathepsin G (CG) expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-κB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase), protein specificity (V8 protease and proteinase K) or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase). We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.

Highlights

Tamm-Horsfall glycoprotein (THP), a renally excreted 80–90 kDa GPI-anchored macromolecule, is an important defense molecule in protecting urinary tract epithelial cells from microbial invasion [1,2].This macromolecule contains approximately 25–35% of florid carbohydrate-side chain structure with abundant sialic acid [3,4]
Molecules including N-acetylneuraminic acid, N-acetylgalactosamine, N-acetylglucosamine and α-methyl-D-mannoside failed to inhibit the mitogenic effect of THP on human mononuclear cells [13]. These results suggest that THP is capable of reacting with surface expressed molecules on different cells, but not through specific CHO containing lectin-like receptors on the cells
Four original findings were derived from the present study: (1) LF and cathepsin G (CG) are the binding molecules of THP expressed on polymorphonuclear neutrophils (PMN) surface; (2) THP transduces PMN activating signals via enhanced ERK 1⁄2, but suppressed p38 MAP kinase phosphorylation; (3) THP does not affect the DNA

Summary

Introduction

Tamm-Horsfall glycoprotein (THP), a renally excreted 80–90 kDa GPI-anchored macromolecule, is an important defense molecule in protecting urinary tract epithelial cells from microbial invasion [1,2]. This macromolecule contains approximately 25–35% of florid carbohydrate-side chain structure with abundant sialic acid [3,4]. THP can bind to the surface membrane of polymorphonuclear neutrophils (PMN) [12], lymphocytes [13], monocytes/macrophages [14] and renal glomerular mesangial cells [13] as a non-specific binder to affect cell functions. The nature of the molecule(s) expressed on PMN surface capable of reacting with THP and the molecular basis of THP-induced

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