MASP-2 Is a Heparin-Binding Protease; Identification of Blocking Oligosaccharides.

Ditmer T Talsma,Mohamed R Daha,Felix Poppelaars,Anita H Meter-Arkema,Stephan P Berger,Pradeep Chopra,Annamaria Naggi,Peter Gál,Geert-Jan Boons,Romain R Vivès,Coen A Stegeman,Wendy Dam,Jacob Van Den Born,The Combat Consortium ,Marc A Seelen

doi:10.3389/fimmu.2020.00732

Abstract

It is well-known that heparin and other glycosaminoglycans (GAGs) inhibit complement activation. It is however not known whether fractionation and/or modification of GAGs might deliver pathway-specific inhibition of the complement system. Therefore, we evaluated a library of GAGs and their derivatives for their functional pathway specific complement inhibition, including the MASP-specific C4 deposition assay. Interaction of human MASP-2 with heparan sulfate/heparin was evaluated by surface plasmon resonance, ELISA and in renal tissue. In vitro pathway-specific complement assays showed that highly sulfated GAGs inhibited all three pathways of complement. Small heparin- and heparan sulfate-derived oligosaccharides were selective inhibitors of the lectin pathway (LP). These small oligosaccharides showed identical inhibition of the ficolin-3 mediated LP activation, failed to inhibit the binding of MBL to mannan, but inhibited C4 cleavage by MASPs. Hexa- and pentasulfated tetrasaccharides represent the smallest MASP inhibitors both in the functional LP assay as well in the MASP-mediated C4 assay. Surface plasmon resonance showed MASP-2 binding with heparin and heparan sulfate, revealing high Kon and Koff rates resulted in a Kd of ~2 μM and confirmed inhibition by heparin-derived tetrasaccharide. In renal tissue, MASP-2 partially colocalized with agrin and heparan sulfate, but not with activated C3, suggesting docking, storage, and potential inactivation of MASP-2 by heparan sulfate in basement membranes. Our data show that highly sulfated GAGs mediated inhibition of all three complement pathways, whereas short heparin- and heparan sulfate-derived oligosaccharides selectively blocked the lectin pathway via MASP-2 inhibition. Binding of MASP-2 to immobilized heparan sulfate/heparin and partial co-localization of agrin/heparan sulfate with MASP, but not C3b, might suggest that in vivo heparan sulfate proteoglycans act as a docking platform for MASP-2 and possibly prevent the lectin pathway from activation.

Highlights

As a part of the innate immune system, complement consists of soluble, and cell bound proteins
Since the lectin pathway (LP) and classical pathway (CP) differ only in their pattern recognition molecules and their serine proteases and we showed that the heparin are unable to inhibit the binding of pattern recognition molecules to pathogen associated molecular patterns, we suggest, based on these data, that the LP is inhibited by heparin via the serine proteases, the MASP enzymes
In this study we tested a library of GAG-related polysaccharides for their complement inhibitory capacity and showed that the LP of complement can be blocked by some heparan sulfates (HS) and heparin- and HS-derived oligosaccharides

Summary

Introduction

As a part of the innate immune system, complement consists of soluble, and cell bound proteins. The CP is initiated by the binding of C1q to IgG or IgM and the LP by pattern recognition molecules binding to carbohydrates of pathogens or self-antigen. This leads to a conformational change and subsequent activation of the associated serine proteases C1r/C1s and MASP-1/MASP-2, respectively. These serine proteases cleave C2 and C4, forming the C4bC2a complex, a C3 convertase which deposits C3b initiating the amplification loop. The AP can be initiated either by auto activation of C3 eventually forming the C3 convertase C3bBb, or by in situ binding of AP stimulator properdin to the cell surface. Formation of the C5 convertase in the end leads to the generation of the C5b-9 membrane attack complex, resulting in cell lysis [1]

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