Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals

Abstract

Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N -glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O -glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O -glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

Deciphering O-glycoprotease substrate preferences with O-Pair Search

“…We note that this approach can also be used to map proteolytic cleavage preferences for any protease, including those like Cathepsin D that digest highly glycosylated O -glycoproteins like mucins but are not professional O -glycoproteases. 70 Finally, we recognize there is currently no search algorithm that allows cleavage to be defined by the presence of specific post-translational modification at a specific residue. Addition of this feature to glycoproteomics search algorithms would greatly improve both O -glycoprotease cleavage motif investigations that seek to better understand their biological functions and also the growing number O -glycoproteomic studies that rely on this emerging class of proteases to generate MS/MS-amenable O -glycopeptides.…”

An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation

“…Indels generated at the sgRNA target site were determined using Synthego ICE indel deconvolution web-based tool ( https://synthego.com ). CTSB , CTSD , and CTSL KO lines were generated using pLentiCRISPRv1 and previously validated ( 55 ).…”

A lysosomal lipid transport pathway that enables cell survival under choline limitation

“…Indels generated at the sgRNA target site were determined using Synthego ICE indel deconvolution web-based tool (https://www.synthego.com). Cathepsin B, D and L knockout lines were generated using pLentiCRISPRv1 and previously validated ( 55 ).…”