The N-Acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis Is a Zinc Metalloenzyme

Daan M.F. van Aalten,Michael A.J. Ferguson,Terry K. Smith,Michael D. Urbaniak,Tunhan Chang,Arthur Crossman

doi:10.1074/jbc.m502402200

Daan M.F. van Aalten, Michael A.J. Ferguson + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m502402200

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Abstract

The de-N-acetylation of N-acetyl-D-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn(2+) > Cu(2+) > Ni(2+) > Co(2+) > Mg(2+). Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.

Highlights

Glycosylphosphatidylinositol (GPI)1 acts as a membrane anchor for a significant proportion of eukaryotic cell surface glycoproteins
Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target
Typanosome Cell-free System Assays—The trypanosome cellfree system converts UDP-[6-3H]GlcNAc to [6-3H]GlcNAc-PI, which is de-N-acetylated by TbGPI12 to give [6-3H]GlcN-PI, which may be partially converted to Man␣1– 4GlcN-PI and later GPI intermediates at the expense of traces of endogenous Dol-P-Man in the cell-free system membrane (Fig. 1A, blank) [32]

Summary

EXPERIMENTAL PROCEDURES

Materials—[9,10-3H]Myristic acid (40.2 Ci/mmol), UDP-[6-3H]GlcNAc (UDP-[3H]GlcNAc, 20 Ci/mmol), and En3HanceTM were purchased from PerkinElmer Life Sciences. The treated membranes were washed, resuspended in 2ϫ incorporation buffer (with or without inhibitor) with 10 mM dithiothreitol, and added to dry UDP-[3H]GlcNAc (1 ␮Ci) and sonicated briefly and incubated at 30 °C for 1 h. The PCR fragment was subcloned into pEGFP-N1 with HindIII and BamHI to give the plasmid pEGFP-rPIG-L. Expression of rPIG-L—The QuikChange procedure (Stratagene) was used to create a silent mutation that removed the internal XhoI site in rPIG-L using the primers 5Ј-CCACCTCTACACCGTCTTTTCGAGATACATGAGCGT-3Ј and 5Ј-ACGCTCATGTATCTCGAAAAGACGGTGTAGAGGTGG-3Ј (mutation in boldface) to give the plasmid pMEEBrPIG-L-FLAG-sXho. The DNA encoding residues 24 –252 of rPIG-L was amplified by PCR from pMEEB-rPIG-L-FLAG-sXho using a 5Ј primer (5Ј-AAACCCATGGATAAGGTTTTAGATTCAGCGGAACGAAT-3Ј) that incorporated an NcoI site (italics) and a six-residue N-terminal sequence (MDKVLD, underlined) and a 3Ј primer (5Ј-TTTTCTCGAGTGCGTAATCAGGGACGTCATAAGGGTAGCCGAGCAACTGCAGC-3Ј) that incorporated a C-terminal HA tag (GYPYDVPDYA, underlined) and a XhoI site (italics). Pictures were made with PyMol [30] and GRASP [31]

RESULTS

32.1 Zinc coordination

DISCUSSION