Purification and biochemical characterization of native ERp29 from rat liver.
ERp29 is a recently characterized resident of the ER (endoplasmic reticulum) lumen that has broad biological significance, being expressed ubiquitously and abundantly in animal cells. As an apparent housekeeper, ERp29 is thought to be a general folding assistant for secretory proteins and to probably function as a PDI (protein disulphide isomerase)-like molecular chaperone. In the present paper, we report the first purification to homogeneity and direct functional analysis of native ERp29, which has led to the unexpected finding that ERp29 lacks PDI-like folding activities. ERp29 was purified 4800-fold in non-denaturing conditions exploiting an unusual affinity for heparin. Two additional biochemical hallmarks that will assist the classification of ERp29 homologues were identified, namely the idiosyncratic behaviours of ERp29 on size-exclusion chromatography (M(r)<globular homodimer) and SDS/PAGE (M(r)>monomeric mass). In contrast with PDI and parallel-purified co-residents (calreticulin, ERp60), native ERp29 lacked classical chaperone, disulphide reductase and isomerase, and calcium-binding activities. In the chaperone assays, ERp29 neither protected substrate proteins against thermal aggregation nor interacted stably with chemically denatured proteins as detected by cross-linking. ERp29 also did not exhibit helper activity toward calreticulin (chaperone) or PDI and ERp60 (disulphide reductase). By refuting long-standing predictions about chaperone activity, these results expose ERp29 as a functionally distinct member of the ER machinery and prompt a revised hypothesis that ERp29 acts as a non-classical folding assistant. The native preparation and biochemical hallmarks established here provide a useful foundation for ongoing efforts to resolve the functional orphan status of ERp29.
Hubbard
Michael J
MJ
Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand. mike.hubbard@unimelb.edu.au
Mangum
Jonathan E
JE
McHugh
Nicola J
NJ
D000818:Animals; D037282:Calreticulin / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D008099:Liver / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D011489:Protein Denaturation; D051381:Rats; D017208:Rats, Wistar
D037282:Calreticulin; C110909:Erp29 protein, rat; D006360:Heat-Shock Proteins