Incorporation of desmocollin‐2 into the plasma membrane requires N‐glycosylation at multiple sites

Abstract

Desmocollin‐2 (DSC2) is a desmosomal protein of the cadherin family. Desmosomes are multiprotein complexes, which are involved in cell adhesion of cardiomyocytes and of keratinocytes. The molecular structure of the complete extracellular domain (ECD) of DSC2 was recently described, revealing three disulfide bridges, four N ‐glycosylation sites, and four O ‐mannosylation sites. However, the functional relevance of these post‐translational modifications for the protein trafficking of DSC2 to the plasma membrane is still unknown. Here, we generated a set of DSC2 mutants, in which we systematically exchanged all N ‐glycosylation sites, O ‐mannosylation sites, and disulfide bridges within the ECD and investigated the resulting subcellular localization by confocal laser scanning microscopy. Of note, all single and double N ‐glycosylation‐ deficient mutants were efficiently incorporated into the plasma membrane, indicating that the absence of these glycosylation sites has a minor effect on the protein trafficking of DSC2. However, the exchange of multiple N ‐glycosylation sites resulted in intracellular accumulation. Colocalization analysis using cell compartment trackers revealed that N ‐glycosylation‐ deficient DSC2 mutants were retained within the Golgi apparatus. In contrast, elimination of the four O ‐mannosylation sites or the disulfide bridges in the ECD has no obvious effect on the intracellular protein processing of DSC2. These experiments underscore the importance of N ‐glycosylation at multiple sites of DSC2 for efficient intracellular transport to the plasma membrane.

Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset

“…They consist of proteins from three different protein families. The cadherins desmoglein-2 and desmocollin-2 (encoded by DSG2 and DSC2 ) are type I transmembrane glycoproteins [ 2 , 3 , 4 ] and mediate the calcium-dependent cell–cell adhesion between cardiomyocytes. Both proteins have extracellular domains consisting of four cadherin domains (EC1–4) and an anchor domain (EA) [ 5 ].…”

Insights Into Genetics and Pathophysiology of Arrhythmogenic Cardiomyopathy

“…Hetero-and homo-or compound heterozygous pathogenic mutations in DSG2 and DSC2 have been described in about 5% of ACM patients [19,[32][33][34]. The majority of DSG2 and DSC2 mutations are localized in the extracellular domains, which consist of four cadherin and an anker domain, carrying several N-glycosylations and O-mannosylations [35,36]. Hetero-and homophilic protein-protein interactions of the desmosomal cadherins are mediated by their first cadherin domains by strand swapping and are calcium-dependent [37].…”

The Double Mutation DSG2-p.S363X and TBX20-p.D278X Is Associated with Left Ventricular Non-Compaction Cardiomyopathy: Case Report

“…HT-1080 cells (German Collection of Microorganisms and Cell Cultures, DSMZ, Brunswick, Germany) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA) and penicillin and streptomycin as previously described [36]. Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) was used for cell transfection according to the manufacturer's instruction.…”