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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.


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Exon 9 confers shedding susceptibility to CADM1 through five non-glycosylatable amino acids.(a) The amino acid sequences of stalk region (between membrane-proximal IgC2 domain and transmembrane domain) of CADM1 variant proteins. The amino acids encoded by exon 8 and exon 9 are colored in magenta and red, respectively. Threonine residues serving as potential O-glycosylation sites are highlighted. An arrowhead indicates the shedding cleavage site of v8/9 CADM1. (b–d) Raw 264.7 cells expressing Halo-tagged v8 CADM1 mutants (b), v8/9 CADM1 mutants (c), or v9 CADM1 mutants (d) were treated with LPS for 60 min, and cell extracts and culture supernatants were subjected to Western blotting. The amino acid sequences of substitution and deletion mutants are indicated above.
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f4: Exon 9 confers shedding susceptibility to CADM1 through five non-glycosylatable amino acids.(a) The amino acid sequences of stalk region (between membrane-proximal IgC2 domain and transmembrane domain) of CADM1 variant proteins. The amino acids encoded by exon 8 and exon 9 are colored in magenta and red, respectively. Threonine residues serving as potential O-glycosylation sites are highlighted. An arrowhead indicates the shedding cleavage site of v8/9 CADM1. (b–d) Raw 264.7 cells expressing Halo-tagged v8 CADM1 mutants (b), v8/9 CADM1 mutants (c), or v9 CADM1 mutants (d) were treated with LPS for 60 min, and cell extracts and culture supernatants were subjected to Western blotting. The amino acid sequences of substitution and deletion mutants are indicated above.

Mentions: Since both scissile bond sequence and sufficient stalk length are considered to be required for shedding susceptibility of membrane proteins, we first examined whether exon 9 encodes the shedding cleavage site of CADM1. After confirming that v8/9 CADM1 is significantly more sensitive to shedding than v8 CADM1 in HEK 293 cells, the cytoplasmic PA-tagged v8/9 CADM1 was highly expressed in HEK 293 cells, and the ~15 kDa membrane-remaining shedding product was affinity purified (Supplementary Fig. 5a,b). The N-terminal sequencing revealed that it starts with Ser-Xaa-Ala-Xaa-Glu-Glu (Supplementary Fig. 5c), indicating that v8/9 CADM1 is cleaved between aspartic acid 374 and serine 375, which reside immediately downstream of the amino acid sequence encoded by exon 9 (Fig. 4a, arrowhead). It was previously reported that ADAM17 characteristically prefers proline residue at P5 position (5 amino acids upstream of the cleavage site)6, which is consistent with the identified cleavage site, suggesting the reliability of our analysis. These observations indicate that not exon 9 but exon 11 encodes the shedding cleavage site of CADM1.


Mechanistic insights into ectodomain shedding: susceptibility of CADM1 adhesion molecule is determined by alternative splicing and O -glycosylation
Exon 9 confers shedding susceptibility to CADM1 through five non-glycosylatable amino acids.(a) The amino acid sequences of stalk region (between membrane-proximal IgC2 domain and transmembrane domain) of CADM1 variant proteins. The amino acids encoded by exon 8 and exon 9 are colored in magenta and red, respectively. Threonine residues serving as potential O-glycosylation sites are highlighted. An arrowhead indicates the shedding cleavage site of v8/9 CADM1. (b–d) Raw 264.7 cells expressing Halo-tagged v8 CADM1 mutants (b), v8/9 CADM1 mutants (c), or v9 CADM1 mutants (d) were treated with LPS for 60 min, and cell extracts and culture supernatants were subjected to Western blotting. The amino acid sequences of substitution and deletion mutants are indicated above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5385562&req=5

f4: Exon 9 confers shedding susceptibility to CADM1 through five non-glycosylatable amino acids.(a) The amino acid sequences of stalk region (between membrane-proximal IgC2 domain and transmembrane domain) of CADM1 variant proteins. The amino acids encoded by exon 8 and exon 9 are colored in magenta and red, respectively. Threonine residues serving as potential O-glycosylation sites are highlighted. An arrowhead indicates the shedding cleavage site of v8/9 CADM1. (b–d) Raw 264.7 cells expressing Halo-tagged v8 CADM1 mutants (b), v8/9 CADM1 mutants (c), or v9 CADM1 mutants (d) were treated with LPS for 60 min, and cell extracts and culture supernatants were subjected to Western blotting. The amino acid sequences of substitution and deletion mutants are indicated above.
Mentions: Since both scissile bond sequence and sufficient stalk length are considered to be required for shedding susceptibility of membrane proteins, we first examined whether exon 9 encodes the shedding cleavage site of CADM1. After confirming that v8/9 CADM1 is significantly more sensitive to shedding than v8 CADM1 in HEK 293 cells, the cytoplasmic PA-tagged v8/9 CADM1 was highly expressed in HEK 293 cells, and the ~15 kDa membrane-remaining shedding product was affinity purified (Supplementary Fig. 5a,b). The N-terminal sequencing revealed that it starts with Ser-Xaa-Ala-Xaa-Glu-Glu (Supplementary Fig. 5c), indicating that v8/9 CADM1 is cleaved between aspartic acid 374 and serine 375, which reside immediately downstream of the amino acid sequence encoded by exon 9 (Fig. 4a, arrowhead). It was previously reported that ADAM17 characteristically prefers proline residue at P5 position (5 amino acids upstream of the cleavage site)6, which is consistent with the identified cleavage site, suggesting the reliability of our analysis. These observations indicate that not exon 9 but exon 11 encodes the shedding cleavage site of CADM1.

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