Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7

Abstract

CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.

Mutations in MFSD8, Encoding a Lysosomal Membrane Protein, Are Associated with Nonsyndromic Autosomal Recessive Macular Dystrophy

“…39,46,47 Studies in gene-targeted mouse embryonic fibroblasts show that MFSD8 is asymmetrically double cleaved for proper function. 48 Analysis of 2 missense mutations demonstrates that the cleavage occurs in the luminal L9 loop, and the proteolytic cleavage is altered by these mutations. 48 The c.1102G>C mutation induces a splice defect and skipping of exon 11, resulting in a truncated protein (p.Lys333Lysfs*3).…”

“…48 Analysis of 2 missense mutations demonstrates that the cleavage occurs in the luminal L9 loop, and the proteolytic cleavage is altered by these mutations. 48 The c.1102G>C mutation induces a splice defect and skipping of exon 11, resulting in a truncated protein (p.Lys333Lysfs*3). Nevertheless, a wild-type transcript lacking exon 11 was seen in a control sample, suggesting that this event also occurs naturally, although more normal transcript (containing exon 11) was observed in the control sample compared with affected individual B:II-1.…”

Proteolytic processing of the neuronal ceroid lipofuscinosis related lysosomal protein CLN5

“…In line with this idea, many soluble lysosomal enzymes including TPP1 (CLN2), CTSD (CLN10), CTSF (CLN13), and acid alpha-glucosidase undergo proteolytic cleavage and maturation in the lysosomes [32–36]. Another NCL polytopic membrane protein, CLN7, has also been observed to undergo cysteine pro-tease-dependent proteolytic cleavage in the lysosome [37]. Further investigation will be needed to understand the importance of CLN5 proteolytic processing and to reveal the function of CLN5.…”

Specific Alleles of CLN7/MFSD8, a Protein That Localizes to Photoreceptor Synaptic Terminals, Cause a Spectrum of Nonsyndromic Retinal Dystrophy

“…It was suggested that some of the missense alleles may represent functional null mutations. 36 Based on the pathogenicity prediction scores of the missense mutations (Supplementary Table S2) and the topology prediction diagram that maps the mutations on the protein sequence (Fig. 5) there appears to be no obvious correlation between the mutations and whether the patient develops vLINCL or nonsyndromic eye disease.…”