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

Abstract

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.Collagen XVII belongs to the family of collagenous transmembrane proteins, which function both as cell membrane receptors and as extracellular matrix components and are involved in a broad spectrum of biological events, ranging from cell adhesion to host defense (1). The group members typically are trimers of identical polypeptides, ␣-chains, which contain an N-terminal intracellular domain, a single transmembrane stretch, and a large extracellular C terminus with one or more collagenous subdomains, i.e. a structure characteristic of type II transmembrane proteins. The ectodomain is shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloprotease domain) family to yield a shorter form of the molecule, which remains stable in the extracellular space after the release (2). As a component of the hemidesmosomes, collagen XVII is involved in the anchorage of stratified epithelia in the skin, the mucous membranes, or the eye to the underlying mesenchyme.Mutations in the collagen XVII gene, COL17A, cause JEB, a genodermatosis characterized by mechanically induced detachment of the epidermis from the dermis. To date, over 30 different COL17A1 mutations have been reported (Human Gene Mutation Database, Cardiff University; Refs. 3 and 4). Most are null mutations resulting in premature termination codons and, subsequently, in nonsense-mediated mRNA d...

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