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J. Biol. Chem., Vol. 279, Issue 33, 34589-34594, August 13, 2004

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Four N-linked Glycosylation Sites in Human Toll-like Receptor 2 Cooperate to Direct Efficient Biosynthesis and Secretion^*

Alexander N. R. Weber¶, Mary A. Morse, and Nicholas J. Gay||

From the RA Pharmacology, RI CEDD, GlaxoSmithKline Medicines Research Centre, Stevenage SG1 2NY, United Kingdom and the Department of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom

Most higher organisms have a system of innate immune defense that is mediated by a group of evolutionarily related, germ line-encoded receptors, so-called Toll-like receptors. In mammals Toll-like receptors signal in response to pathogen-associated microbial structures. For example, Toll-like receptor 2 appears to mediate responses to bacterial peptidoglycan and acylated lipoproteins and Toll-like receptor 4 to bacterial lipopolysaccharide. However, the structural principles that underlie recognition of these structures are poorly understood. Toll-like receptors have leucine-rich repeats in their extracellular domains and are thus believed to adopt solenoid structures, similar to that found in platelet glycoprotein Ib. Additionally, all Toll-like receptors contain N-linked glycosylation consensus sites, and Toll-like receptor 4 requires glycosylation for function. Toll-like receptor glycosylation is also likely to influence receptor surface representation, trafficking, and pattern recognition. Using circular dichroism spectroscopy, we show here that purified human Toll-like receptor 2 and 4 proteins have secondary structure contents similar to glycoprotein Ib. We have also analyzed where consensus glycosylation sites are located in the extracellular domains of other human Toll-like receptors. We found that there are significant differences in the location and degree of conservation between sites in different Toll-like receptors. Using site-directed mutagenesis, we have found that in Toll-like receptor 2 extracellular domain all four predicted glycosylation sites are substituted, although one site is inefficiently core-glycosylated and its removal drastically affects secretion. The remaining Toll-like receptor 2 glycosylation sites also contribute to efficient protein secretion, albeit to a lesser degree.

Received for publication, April 6, 2004 , and in revised form, May 25, 2004.

^* 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 a supplemental figure and a supplemental table.

^¶ Supported by a studentship from GlaxoSmithKline and St. John's College, Cambridge.

^|| To whom correspondence should be addressed. Tel.: 44-1223-333626; Fax: 44-1223-766002; E-mail: njg11{at}mole.bio.cam.ac.uk.

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