Expression and specificity of human G(M2) activator protein

Yun Wu Yan Yun Wu, J. M. Lockyer, E. Sugiyama, N. V. Pavlova, Y. T. Li, S. C. Li

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61 Scopus citations

Abstract

The cDNA encoding G(M2) activator was expressed in the Escherichia coli/pT7-7 system. The yield of the G(M2) activator with greater than 99% purity was about 3 mg per liter culture. The recombinant G(M2) activator was found to be as active as that isolated from human kidney. The availability of the recombinant G(M2) activator enabled us to critically examine the specificity of this activator protein. Our results show that the specificity of G(M2) activator is not as strict as that reported previously. Although G(M2) activator stimulates most efficiently the degradation of G(M2) carried out by β-N-acetylhexosaminidase A (Hex A), this activator also stimulates the following reactions: (a) conversion of G(M2) to G(A2) by clostridial sialidase; (b) hydrolysis of GalNAc from dipalmitoylphosphatidylethanolamine·II3NeuAcGgOse3 by Hex A; and (c) liberation of Gal from G(M1) by β-galactosidase at a high activator concentration. Thus, this activator does not differentiate between G(M2) and dipalmitoylphosphatidylethanolamine·II3NeuAcGgOse3 or between Hex A and clostridial sialidase. The micellar forms of G(D2) and GalNAc-G(D1a) were found to be more readily hydrolyzed by Hex A than G(M2) in the absence of G(M2) activator. Our results also show that saposin B can enhance the stimulatory activity of G(M2) activator, but it cannot promote the stimulatory activity of sodium taurodeoxycholate. Taken together, our results suggest that the mechanism of action of G(M2) activator is different from saposin B, and the action of G(M2) activator is more than to solubilize lipid substrates. The effectiveness of G(M2) activator in stimulating the hydrolysis of G(M2) may be due to its ability to recognize the specific trisaccharide structure of the G(M2) epitope, GalNAcβ1 → 4(NeuAcα2 → 3)Gal-, and to modify the GalNAc-NeuAc interaction in this structure.

Original languageEnglish (US)
Pages (from-to)16276-16283
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number23
StatePublished - 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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