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J. Biol. Chem., Vol. 281, Issue 28, 19545-19560, July 14, 2006

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Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 -N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core^*

Noboru Tomiya¹, Someet Narang, Jung Park^¶, Badarulhisam Abdul-Rahman, One Choi^||, Sundeep Singh^¶, Jun Hiratake^**, Kanzo Sakata^**, Michael J. Betenbaugh, Karen B. Palter^¶, and Yuan C. Lee

From the Departments of Biology and Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, the ^¶Department of Biology, Temple University, Philadelphia, Pennsylvania 19122, the ^||Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-gu, Taejon 305-701, Korea, and the ^**Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a -N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a -N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the (1,2)-linked N-acetylglucosamine from the Man(1,3) branch compared with the Man(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal -N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing -N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

Received for publication, April 6, 2006 , and in revised form, May 2, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ183186 and DQ183187. The nucleotide sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession number Q3LS76.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and Tables SI and SII.

¹ To whom correspondence should be addressed: Dept. of Biology, The Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218. Tel.: 410-516-7322; Fax: 410-516-8716; E-mail: ntomiya1{at}jhu.edu.

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