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J. Biol. Chem., Vol. 282, Issue 30, 21934-21944, July 27, 2007

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Heparan Sulfate Degradation Products Can Associate with Oxidized Proteins and Proteasomes^*

From the Department of Experimental Medical Science, Section of Neuroscience, Lund University, Biomedical Centre A13, SE-221 84, Lund, Sweden

The S-nitrosylated proteoglycan glypican-1 recycles via endosomes where its heparan sulfate chains are degraded into anhydromannose-containing saccharides by NO-catalyzed deaminative cleavage. Because heparan sulfate chains can be associated with intracellular protein aggregates, glypican-1 autoprocessing may be involved in the clearance of misfolded recycling proteins. Here we have arrested and then reactivated NO-catalyzed cleavage in the absence or presence of proteasome inhibitors and analyzed the products present in endosomes or co-precipitating with proteasomes using metabolic radiolabeling and immunomagnet isolation as well as by confocal immunofluorescence microscopy. Upon reactivation of deaminative cleavage in T24 carcinoma cells, [³⁵S]sulfate-labeled degradation products appeared in Rab7-positive vesicles and co-precipitated with a 20 S proteasome subunit. Simultaneous inhibition of proteasome activity resulted in a sustained accumulation of degradation products. We also demonstrated that the anhydromannose-containing heparan sulfate degradation products are detected by a hydrazide-based method that also identifies oxidized, i.e. carbonylated, proteins that are normally degraded in proteasomes. Upon inhibition of proteasome activity, pronounced colocalization between carbonyl-staining, anhydro-mannose-containing degradation products, and proteasomes was observed in both T24 carcinoma and N2a neuroblastoma cells. The deaminatively generated products that co-precipitated with the proteasomal subunit contained heparan sulfate but were larger than heparan sulfate oligosaccharides and resistant to both acid and alkali. However, proteolytic degradation released heparan sulfate oligosaccharides. In Niemann-Pick C-1 fibroblasts, where deaminative degradation of heparan sulfate is defective, carbonylated proteins were abundant. Moreover, when glypican-1 expression was silenced in normal fibroblasts, the level of carbonylated proteins increased raising the possibility that deaminative heparan sulfate degradation is involved in the clearance of misfolded proteins.

Received for publication, February 8, 2007 , and in revised form, May 10, 2007.

^* The work was supported by grants from the Swedish Science Council (VR-M), the Bergvall, Crafoord, Hedborg, Jeansson, Kock, Segerfalk, Zoega, and Österlund Foundations, and the Medical Faculty of Lund University. 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 may be addressed. Tel.: 46-46-222-8573; Fax: 46-46-222-0615; E-mail: katrin.mani{at}med.lu.se. ² To whom correspondence may be addressed. Tel.: 46-46-222-8573; Fax: 46-46-222-0615; E-mail: lars-ake.fransson{at}med.lu.se.

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