Over-expression of a functionally active human GM2-activator protein in Escherichia coli

Abstract

The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.

Biosynthesis, Processing, and Intracellular Transport of GM2 Activator Protein in Human Epidermal Keratinocytes

“…After Klima et al (12) had found that recombinant, nonglycosylated G M2 AP is effectively endocytosed by human fibroblasts, the question arose whether its intracellular transport would also be at least partially independent of M6P or other carbohydratelinked signals. Several different lines of evidence demonstrated that such a pathway must exist in hEKs.…”

“…Although the targeting of soluble lysosomal proteins usually involves the mannose-6-phosphate receptor system (reviewed in Ref. 11), it was found that recombinant, nonglycosylated G M2 AP is effectively endocytosed by AB variant fibroblasts (12). Thus, at least the endocytosis of G M2 AP can occur independently of M6P residues and this raises the question, how far its intracellular transport would depend on the M6P pathway.…”

GM2 activator protein inhibits platelet activating factor signaling in rats

“…All groups received analgesia and anesthesia; the first by subcutaneous (s.c.) injection 30 min prior to LPS/PAF; and the second by a mixture of anesthetic and oxygen/nitrous oxide. Human recombinant GM2AP (rGM2AP) was prepared as reported [12,21] sterile filtered and stored at 4 °C. Human rGM2AP was given either once (1 h before the PAF/LPS injection) or twice (1 h before and 1 h after) for treatment/prevention of NEC like-lesions at 1.2 mg/kg.…”

Complete localization of disulfide bonds in GM2 activator protein

“…Its amino acid sequence has been deduced from the cDNA structure and has been confirmed by Edman degradation (Furst et al, 1990). The protein has been made available in large amounts by recombinant expression and refolding from Escherichia coli inclusion bodies (Klima et al, 1993;Wu et al, 1996a), and recently from expression in the baculovims system without the necessity of refolding (T. Lemm, 0. Bartelsen, & K. Sandhoff, unpubl.…”