Attenuation of 19-9 Antigen Secretion in Human Colorectal Carcinoma Cell Cultures by Transfection with cDNA Encoding Novel ADP-Ribosylation Factor-like Proteins

We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Δ) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning. This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl resid...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Archives of biochemistry and biophysics 1998-02, Vol.350 (2), p.145-156
Hauptverfasser: Eboue, D., Icard-Liepkalns, C., Beringer, T.M., Liepkalns, V.A.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Δ) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning. This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl residues, 4 functioning consensus sequence GTP binding sites, and 184 total aminoacyl residues. An ARL cDNA was also constructed deleting the codon for the N-terminal glycyl moiety. The resulting cell clones were shown by Northern blots to overexpress ARL mRNA. Electron microscopy–immunocytochemistry also indicated the overexpression of ARL granules subcellularly. Secretion of the tumor-associated 19-9 antigen into apical medium was decreased 3- to 5-fold and the secretion of TCA/PTA precipitable3H-labeled glycoprotein was decreased by 34% in clone SW1116(ARL184)Δ. Western blot analyses of cell homogenates and media were in agreement with the secretion assays and showed a diminution of 170–200 kDa, 19-9, antigenicity in transfected cells and their media. Apical secretion of 19-9 antigen was diminished 14-fold in cells, SW1116 (ARL184)α, transfected with the complete ARL cDNA sequence, suggesting that the glycyl moiety may be required for maximal abatement. However, incorporation of label from [3H]myristate into 22-kDa bands of NP-40 extracts and ARL-antigenic molecules of parent cells was 3-fold greater than that in samples from the two transfectants; thus the transfected cells may not myristylate the overexpressed ARL efficiently. Notwithstanding the N-terminal glycyl moiety undergoing some other modification, we conclude that overexpression of this ARL is sufficient to generate a 19-9-deficient phenotype. These ARLs may eventually disrupt terminal oligosaccharide glycosylation, resulting in an apparent diminished exocytosis of 19-9 glycoprotein carriers by transfected and cloned cells.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.1997.0493