Heterodimerization of the Sialidase NEU1 with the Chaperone Protective Protein/Cathepsin A Prevents Its Premature Oligomerization

Erik J Bonten,Yvan Campos,Viateslav Zaitsev,Amanda Nourse,Brett Waddell,William Lewis,Garry Taylor,Alessandra D'Azzo

doi:10.1074/jbc.m109.031419

Erik J Bonten, Yvan Campos + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.031419

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Abstract

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.

Highlights

To better understand the mechanism of NEU1 activation, we examined the ability of purified precursor protective protein/cathepsin A (PPCA) and mature PPCA to activate NEU1 in solution
Using purified proteins in a cell-free system, we showed that precursor PPCA and mature, active PPCA are both capable of activating NEU1 in a concentration-dependent manner
NEU1 bound to a deletion mutant of PPCA that corresponded to the 20-kDa C-terminal part of PPCA; yet again, no high Mr complex was formed

Summary

EXPERIMENTAL PROCEDURES

Cell Culture, Antibodies, and Recombinant Proteins—Fibroblasts from a patient with early infantile galactosialidosis were deposited in the European Cell Bank (Rotterdam, The Netherlands). Purified insect cell-expressed PPCA and NEU1 proteins were covalently attached to a CM-5 chip (Biacore) using standard amine coupling methods in 10 mM sodium acetate at pH 5.5. Binding of NEU1, PPCA, or NEU1 synthetic 15-mer peptides was measured by flowing protein or peptides in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20 (HBS-EP buffer, Biacore) at 20 ␮l/min in sequence through the reference and protein-containing flow cells at 25 °C. Analytical Ultracentrifugation—Purified insect cell-expressed NEU1 and PPCA were dialyzed against the ultracentrifugation buffers, 50 mM sodium acetate, pH 5, or phosphatebuffered saline, 100 mM NaCl, pH 7.0, and subjected to sedimentation velocity in a ProteomeLab XL-I analytical ultracentrifuge with an eight-hole Beckman An-50 rotor and cells containing sapphire windows and charcoal-filled Epon doublesector center pieces (Beckman Coulter, Fullerton, CA). Attempts were undertaken to discriminate between various models using the protein-protein docking servers ClusPro [50], RosettaDock [51, 52], and Gramm-X [53, 54], with the knowledge-score algorithms based on surface complementarity, energy optimization, and fast Fourier transformation methodologies, respectively

RESULTS

Protein complex kDa

DISCUSSION