N-glycosylation of MDA-7/IL-24 is dispensable for tumor cell-specific apoptosis and "bystander" antitumor activity.
Biochemical and genetic mutation-based analyses confirm that the MDA-7/IL-24 protein can induce transformed cell-specific apoptosis through a mechanism involving endoplasmic reticulum (ER) stress-associated pathways. Covalent modifications by N-linked glycans in the ER contribute to the conformational maturation and biological functions of many proteins. Because MDA-7/IL-24 is a glycosylated protein, we investigated the role of glycosylation in mediating the specific biological and "bystander" antitumor activities of this cytokine. An adenovirus vector expressing a nonsecreted and nonglycosylated version of MDA-7/IL-24 protein was generated via deletion of its signal peptide and point mutations of its three N-glycosylated sites. In this study, we showed that this intracellular nonglycosylated protein was as effective as wild-type MDA-7/IL-24 protein in inducing apoptosis in multiple tumor cell lines. Both constructs (a) displayed transformed cell specificity and localization to the ER compartment, (b) mediated apoptosis through JAK/STAT-independent and p38(MAPK)-dependent pathways, (c) induced sustained ER stress as evidenced by expression of ER stress markers (BiP/GRP78, GRP94, XBP-1, and eIF2alpha), and (d) generated proteins that physically interacted with BiP/GRP78. Additionally, an expression construct containing the mda-7/IL-24 signal peptide linked to the mutated nonglycosylated mda-7/IL-24 gene retained the ability to induce bystander antitumor activity. These studies reveal that MDA-7/IL-24 glycosylation is not mandatory for inducing cell death or bystander activities in different cancer cells, providing new insights into the mechanism by which MDA-7/IL-24 induces apoptosis and ER stress.
Sauane
Moira
M
Department of Urology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York 10032, USA.
Gupta
Pankaj
P
Lebedeva
Irina V
IV
Su
Zao-Zhong
ZZ
Sarkar
Devanand
D
Randolph
Aaron
A
Valerie
Kristoffer
K
Gopalkrishnan
Rahul V
RV
Fisher
Paul B
PB
D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms; D024201:Bystander Effect; D016923:Cell Death; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D005434:Flow Cytometry; D005818:Genetic Engineering; D006031:Glycosylation; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D007378:Interleukins / Q000235:genetics* / Q000378:metabolism*; D000099180:Interleukin-24
D018076:DNA, Complementary; D000091342:Endoplasmic Reticulum Chaperone BiP; D051547:Heme Oxygenase-1; D007378:Interleukins; D000099180:Interleukin-24; C000717247:HSPA5 protein, human