Abstract
Ribosomal protein S3 (rpS3) is a 243 amino acid component of the 40S ribosomal small subunit. It has multiple roles in translation and extra-ribosomal functions like apoptosis and DNA repair. RpS3 is secreted only in cancer cell lines. Presently, mass spectrometry analysis revealed rpS3 to be glycosylated at the Asn165 residue. A point mutation at this residue decreased secretion of rpS3 in cancer cell lines. Secretion was also inhibited by the endoplasmic reticulum (ER)-Golgi transport inhibitor Brefeldin A and by Tunicamycin, an inhibitor of N-linked glycosylation. N-linked glycosylation of rpS3 was confirmed as necessary for rpS3 secretion into culture media via the ER-Golgi dependent pathway. RpS3 bound to Concanavalin A, a carbohydrate binding lectin protein, while treatment with peptide-N-glycosidase F shifted the secreted rpS3 to a lower molecular weight band. In addition, the N165G mutant of rpS3 displayed reduced secretion compared to the wild-type. An in vitro binding assay detected rpS3 homodimer formation via the N-terminal region (rpS3:1–85) and a middle region (rpS3:95–158). The results indicate that the Asn 165 residue of rpS3 is a critical site for N-linked glycosylation and passage through the ER-Golgi secretion pathway.
Highlights
Ribosomal protein S3 is a constituent of the 40 S ribosomal small subunit, which functions in translation
To confirm whether the secretion of Ribosomal protein S3 (rpS3) was regulated by pathway of endoplasmic reticulum (ER) to Golgi or Golgi to ER, HT1080 cells were treated with Brefeldin A as an ER-Golgi transport inhibitor (Figure 1A and 1B) [15] or Monensin as a Golgi-ER transport inhibitor (Figure 1C and D) [16]
To quantify Enzyme-linked immunosorbent assay (ELISA) assay of secreted rpS3, we performed ELISA assay with recombinant rpS3 protein (Supplementary Figure 1A and 1B)
Summary
Ribosomal protein S3 (rpS3/RPS3/Ribosomal Protein S3) is a constituent of the 40 S ribosomal small subunit, which functions in translation. RpS3 interacts with nm23-H1, which acts as a suppressor of metastasis in certain human tumors and prevents the invasive potential in HT1080 cells [5]. RpS3 is overexpressed in colorectal cancer cells, suggesting that the level of rpS3 may be related to tumorigenesis [6]. The level of rpS3 secretion was prominently increased in highly malignant cells when compared to normal parent cells [7]. This suggests that secreted rpS3 may be a putative marker for malignant tumors
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