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J. Biol. Chem., Vol. 282, Issue 20, 14942-14951, May 18, 2007

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Regulation of Heparan Sulfate 6-O-Sulfation by -Secretase Activity^*

Naoko Nagai, Hiroko Habuchi, Shinobu Kitazume, Hidenao Toyoda^¶, Yasuhiro Hashimoto, and Koji Kimata¹

From the Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan, Glyco-chain Functions Laboratory, RIKEN Frontier Research System, Hirosawa, Wako-shi, Saitama 351-1098, Japan, and the ^¶Laboratory of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522, Japan

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable -secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of -secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent -secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by -secretase via an indirect mechanism.

Received for publication, November 17, 2006 , and in revised form, January 22, 2007.

^* This work was supported in part by grants-in-aid for scientific research (B) from the Japan Society for the Promotion of Science, grants-in-aid for scientific research (C) from the Japan Society for the Promotion of Science (17570099), a grant-in-aid for scientific research on priority areas 14082206 and 18770119 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a special research fund from Seikagaku Corp. 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.

¹ To whom correspondence should be addressed: Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan. Tel.: 81-52-264-4811 (ext. 2088); Fax: 81-561-63-3532; E-mail: kimata{at}amugw.aichi-med-u.ac.jp.

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