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J. Biol. Chem., Vol. 281, Issue 40, 30260-30268, October 6, 2006

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C-terminal Truncation Impairs Glycosylation of Transmembrane Collagen XVII and Leads to Intracellular Accumulation^*

Claus-Werner Franzke, Cristina Has, Carsten Schulte, Laura Huilaja^¶, Kaisa Tasanen^¶, Monique Aumailley^||, and Leena Bruckner-Tuderman¹

From the Department of Dermatology, University Medical Center Freiburg, 79104 Freiburg, Germany, the Department of Biochemistry, Medical Faculty, University of Cologne, 50923 Cologne, Germany, the ^¶Department of Dermatology, University of Oulu and Clinical Research Center, Oulu University Hospital, 90014 Oulu, Finland, and the ^||Center for Molecular Medicine Cologne, University of Cologne, 50923 Cologne, Germany

Collagen XVII, a type II transmembrane protein in hemidesmosomes, is involved in the anchorage of stratified epithelia to the underlying mesenchyme. Its functions are regulated by ectodomain shedding, and its genetic defects lead to epidermal detachment in junctional epidermolysis bullosa (JEB), a heritable skin fragility syndrome, but the molecular disease mechanisms remain elusive. Here we used a spontaneously occurring homozygous COL17A1 deletion mutant in JEB to discern glycosylation of collagen XVII. The mutation truncated the distal ectodomain and positioned the only N-glycosylation site 34 amino acids from the newly formed C terminus, which impaired efficient N-glycosylation. Immunofluorescence staining of authentic JEB keratinocytes and of COS-7 cells transfected with the mutant indicated intracellular accumulation of collagen XVII precursor molecules. Cell surface biotinylation and quantification of ectodomain shedding demonstrated that only about 15% of the truncated collagen XVII reached the cell surface. The cell surface-associated molecules were N-glycosylated in a normal manner, in contrast to the molecules retained within the cells, indicating that N-glycosylation of the ectodomain is required for targeting of collagen XVII to the plasma membrane and that reduced accessibility of the N-glycosylation site negatively regulates this process. Functional consequences of the strong reduction of collagen XVII on the cell surface included scattered deposition of cell adhesion molecule laminin 5 into the extracellular environment and, as a consequence of faulty collagen XVII-laminin ligand interactions, aberrant motility of the mutant cells.

Received for publication, May 10, 2006 , and in revised form, July 27, 2006.

^* This work was supported in part by grant Br1475/6-3 and SFB620-TPC5 from the DFG (Deutsche Forschungsgemeinschaft), the European Union Concerted Action "Geneskin," the Network "Epidermolysis bullosa" (projects 5 and 9) from the Federal Ministry for Education and Research (BMBF), DFG grant AU86/5-3, and grants from the Academy of Finland, the Alexander von Humboldt Foundation, the René Touraine Foundation, Finnish-Norwegian Medical Foundation, Finnish Medical Foundation, Finnish National Graduate School of Clinical Investigation, and Oulu University Hospital. 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: Dr. Dept. of Dermatology, University Medical Center Freiburg, Hauptstr. 7, 79104 Freiburg, Germany. Tel.: 49-761-270-6716; Fax: 49-761-270-6936; E-mail: bruckner_tuderman{at}haut.ukl.uni-freiburg.de.

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