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Mechanistic insights into ectodomain shedding: susceptibility of CADM1 adhesion molecule is determined by alternative splicing and O -glycosylation

View Article: PubMed Central - PubMed

ABSTRACT

Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation.

No MeSH data available.


Related in: MedlinePlus

Shedding susceptibility of CADM1 is determined by both alternative splicing and O-glycosylation.Schematic diagram of the structure of stalk region of CADM1 variant proteins and their shedding susceptibility. Orange, IgV domain; yellow, IgC2 domain; magenta, amino acids encoded by exon 8; red, amino acids encoded by exon 9; broken lines, O-glycans; blue scissors, ADAM17.
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f6: Shedding susceptibility of CADM1 is determined by both alternative splicing and O-glycosylation.Schematic diagram of the structure of stalk region of CADM1 variant proteins and their shedding susceptibility. Orange, IgV domain; yellow, IgC2 domain; magenta, amino acids encoded by exon 8; red, amino acids encoded by exon 9; broken lines, O-glycans; blue scissors, ADAM17.

Mentions: We further elucidate how the small alternative exon, exon 9, confers shedding susceptibility to CADM1 in the presence of interfering O-glycans. Among 11 amino acids encoded by exon 9, five C-terminal non-glycosylatable amino acids which reside immediately upstream of the shedding cleavage site are indispensable for conferring shedding susceptibility, and these amino acids can be substituted to alanine residues without affecting the shedding susceptibility of exon 9-containing CADM1 variants. These results indicate that “a stretch of five non-glycosylated amino acids” is sufficient to confer shedding susceptibility, that is, exon 9 confers shedding susceptibility to CADM1 by increasing the distance between interfering O-glycans and the shedding cleavage site (Fig. 6). Therefore, we would like to propose here that the term “stalk length”, a determinant of shedding susceptibility, should be redefined as the length between the cell surface and membrane-proximal extracellular domain “or membrane-proximal O-glycosylated amino acid”. Applying this new concept of stalk length, our results further indicate that as few as 5-amino-acid extension of stalk length can switch the shedding susceptibility of CADM1. These observations suggest that membrane proteins have their own “threshold stalk length”, and only membrane proteins having stalk region longer than that are susceptible to shedding. In other words, this study emphasizes the role of stalk length in determining shedding susceptibility.


Mechanistic insights into ectodomain shedding: susceptibility of CADM1 adhesion molecule is determined by alternative splicing and O -glycosylation
Shedding susceptibility of CADM1 is determined by both alternative splicing and O-glycosylation.Schematic diagram of the structure of stalk region of CADM1 variant proteins and their shedding susceptibility. Orange, IgV domain; yellow, IgC2 domain; magenta, amino acids encoded by exon 8; red, amino acids encoded by exon 9; broken lines, O-glycans; blue scissors, ADAM17.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385562&req=5

f6: Shedding susceptibility of CADM1 is determined by both alternative splicing and O-glycosylation.Schematic diagram of the structure of stalk region of CADM1 variant proteins and their shedding susceptibility. Orange, IgV domain; yellow, IgC2 domain; magenta, amino acids encoded by exon 8; red, amino acids encoded by exon 9; broken lines, O-glycans; blue scissors, ADAM17.
Mentions: We further elucidate how the small alternative exon, exon 9, confers shedding susceptibility to CADM1 in the presence of interfering O-glycans. Among 11 amino acids encoded by exon 9, five C-terminal non-glycosylatable amino acids which reside immediately upstream of the shedding cleavage site are indispensable for conferring shedding susceptibility, and these amino acids can be substituted to alanine residues without affecting the shedding susceptibility of exon 9-containing CADM1 variants. These results indicate that “a stretch of five non-glycosylated amino acids” is sufficient to confer shedding susceptibility, that is, exon 9 confers shedding susceptibility to CADM1 by increasing the distance between interfering O-glycans and the shedding cleavage site (Fig. 6). Therefore, we would like to propose here that the term “stalk length”, a determinant of shedding susceptibility, should be redefined as the length between the cell surface and membrane-proximal extracellular domain “or membrane-proximal O-glycosylated amino acid”. Applying this new concept of stalk length, our results further indicate that as few as 5-amino-acid extension of stalk length can switch the shedding susceptibility of CADM1. These observations suggest that membrane proteins have their own “threshold stalk length”, and only membrane proteins having stalk region longer than that are susceptible to shedding. In other words, this study emphasizes the role of stalk length in determining shedding susceptibility.

View Article: PubMed Central - PubMed

ABSTRACT

Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation.

No MeSH data available.


Related in: MedlinePlus