Characterization of Secondary Structure and Lipid Binding Behavior of N-terminal Saposin Like Subdomain of Human Wnt3a

Overview

Journal Arch Biochem Biophys

Publisher Elsevier

Specialties Biochemistry
Biophysics

Date 2017 Jul 30

PMID 28754322

Authors

Aparna Krishnamoorthy

Andrzej Witkowski

Jesse J Tran

Paul M M Weers

Robert O Ryan

Affiliations

Soon will be listed here.

Abstract

Wnt signaling is essential for embryonic development and adult homeostasis in multicellular organisms. A conserved feature among Wnt family proteins is the presence of two structural domains. Within the N-terminal (NT) domain there exists a motif that is superimposable upon saposin-like protein (SAPLIP) family members. SAPLIPs are found in plants, microbes and animals and possess lipid surface seeking activity. To investigate the function of the Wnt3a saposin-like subdomain (SLD), recombinant SLD was studied in isolation. Bacterial expression of this Wnt fragment was achieved only when the core SLD included 82 NT residues of Wnt3a (NT-SLD). Unlike SAPLIPs, NT-SLD required the presence of detergent to achieve solubility at neutral pH. Deletion of two hairpin loop extensions present in NT-SLD, but not other SAPLIPs, had no effect on the solubility properties of NT-SLD. Far UV circular dichroism spectroscopy of NT-SLD yielded 50-60% α-helix secondary structure. Limited proteolysis of isolated NT-SLD in buffer and detergent micelles showed no differences in cleavage kinetics. Unlike prototypical saposins, NT-SLD exhibited weak membrane-binding affinity and lacked cell lytic activity. In cell-based canonical Wnt signaling assays, NT-SLD was unable to induce stabilization of β-catenin or modulate the extent of β-catenin stabilization induced by full-length Wnt3a. Taken together, the results indicate neighboring structural elements within full-length Wnt3a affect SLD conformational stability. Moreover, SLD function(s) in Wnt proteins appear to have evolved away from those commonly attributed to SAPLIP family members.

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