Dysregulation of the Expression of Asparagine-Linked Glycosylation 13 Short Isoform 2 Affects Nephrin Function by Altering Its N-Linked Glycosylation.
BACKGROUND
N-linked glycosylation, which is a post-translational modification process, plays an important role in protein folding, intracellular trafficking and membrane targeting, as well as in regulating the protein function. Recently, we identified a missense variant (p.T141L) in the short isoform 2 of the X-linked gene asparagine-linked glycosylation 13 (ALG13-is2), which segregated with focal segmental glomerulosclerosis and PCCD in a large Australian pedigree; however, any evidence of its pathogenicity was demonstrated. ALG13 gene encodes, through alternative splicing, 2 glycosyltransferase isoforms, which catalyse the second sugar addition of the highly conserved oligosaccharide precursor in the endoplasmic reticulum (ER). Mutations in the long isoform 1 were associated with epilepsy.


RESULTS
Here, we show a different expression of the 2 isoforms depending on the tissue. Specifically, the long isoform is highly expressed in lungs, ovaries, testes, cerebellum, cortex, retina, pituitary gland, and olfactory bulbs, while the short isoform is highly expressed in mouse podocytes and in human podocyte cell lines, at both mRNA and protein levels. The silencing of ALG13-is2 by specific siRNAs induces an altered N-linked glycosylation pattern of nephrin, as demonstrated by the presence of an additional immunostaining band of about 130 kD. In knock-down cells, immunofluorescence analysis shows perturbed organization of the cytoskeleton and altered localization of nephrin on the cellular membrane. We also demonstrated that the altered pattern of N-linked glycosylation induces an over-expression of binding immunoglobulin protein and calreticulin, suggesting ER stress.


CONCLUSIONS
These results provide preliminary evidence that ALG13-is2 could be an important modifier of renal filtration defects.
Institute of Genetics and Biophysics "Adriano Buzzati-Traverso," National Research Council of Italy, Naples, Italy.
Teresa
T
Esposito
Giovanni
G
De Stefano
Mafalda Giovanna
MG
Reccia
Ilaria
I
Di Lorenzo
Filomena
F
Napolitano
Francesco
F
Scalabrì
Alessia
A
Lombardi
Moin A
MA
Saleem
Lyn R
LR
Griffiths
Fernando
F
Gianfrancesco
D000818:Animals; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007536:Isomerism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics; D050199:Podocytes / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014018:Tissue Distribution
D008565:Membrane Proteins; C113267:nephrin; C583889:ALG13 protein, human; D017351:N-Acetylglucosaminyltransferases
Focal segmental glomerulosclerosis; Gene expression; Genetic diseases; Neuropathy (epilepsy); Protein glycosylation; Urinary podocytes