Novel function of tetraspanin‐interacting protein IGSF3 in the regulation of glycosphingolipid metabolism

Abstract

RationaleWe have previously identified in a patient with severe emphysema with stable chromosomal translocation a disruption in the gene encoding for Immunoglobulin Superfamily, Member 3 (Igsf3) accompanied by low levels of this membrane‐anchored protein. We set out to identify the function of Igsf3, which is predicted to interact with other membrane proteins, tetraspanins. cDNA microarray analyses identified that loss of IGSF3 significantly altered gene expression in the glycosphingolipid (GSL) pathway. Since GLS interaction with tetraspanins at the plasma membrane (dubbed as glycosynapse microdomain) inhibits integrin‐mediated cell functions, we hypothesized that Igsf3 is required for the glycosynapse control of cell motility.MethodsTetraspanin‐enriched microdomains (TEMs) were isolated using density gradient‐ultracentrifugation from human bronchial airway epithelial cells (Beas2b) and from primary immortalized human lung lymphocytes isolated from the consenting subject. Beas2b were transduced or transfected with adenoviral‐ and mammalian‐ IGSF3 plasmid vectors; or with siRNA and lentiviral‐shRNA. GSL were measured by LC‐MS/MS and cell migration and proliferation were assessed by standard techniques.ResultsCo‐immunoprecipitation studies identified that within TEMs, Igsf3 colocalized with the tetraspanins CD9, CD81, CD82, and CD151, as well as with integrinb1 (Itgb1). As expected, when compared to other cellular fractions, TEMs were enriched in cholesterol, ceramides, and glycosylceramides (GC). IGSF3 knockdown significantly increased C16‐GC and C22‐GC (by 5.2‐ and 6.2‐ fold, respectively; p<0.0001), whereas IGSF3 overexpression tended to decrease glycosylceramide levels. Loss of Igsf3 by either siRNA or shRNA significantly delayed wound repair in a cell monolayer scratch assay (p<0.0001) and inhibited cell migration (p<0.03), but did not affect cell proliferation.ConclusionsIgsf3 controls glucosylceramide abundance in TEMs and is required for cell migration. While the mechanism by which Igsf3 regulates the GSL metabolism remains to be investigated, our results suggest that Igsf3 may be an important component of the glycosynapse with a functional role in cellular motility and injury repair.Support or Funding InformationWollowick Chair in Respiratory Research; Medresearch AEF_Schweitzer