• Journal of Life Science
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• v.8 no.2
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• pp.189-196
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• 1998

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various animal cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. A similar notif was observed in the sequence of the mouse lymphocyte ADP-ribosyltransferase (Yac-s). Yac-2 has significant NAD glycohydrolase activity as well as ADP-ribosyltransferase activity. To verify the role of the Glu-rich motif in Yac-2, site-directed mutagenesis was performed. Mutants E220Q, E220A, E222A were inactive for ADP-ribosyltransferase activity. For NAD glycohydrolase activity, E220A, E222Q, and E222A were inactive. In contrast, E220Q was active as wild-type. Thus, Glu-220 and Glu-222 in Yac-2 are critical for ADP-ribosyltransferase and NAD glycohydrolase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the Yac-2 enzyme activity.

• Journal of Life Science
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• v.8 no.5
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• pp.486-491
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• 1998

Previously, we have cloned and characterized two ADP-ribosyltransferases (Yac-1 and Yac-2) from mouse Iym-phocyte. Yac-2 transferase contains significant NAD glycohydrolase activity as well as ADP-ribosyltransferase acti-vity. Yac-2 has an arginine at position 221 between two conserved glutamic acids. To investigate the significance of Arg-221 on enzyme activities, Arg-221 was mutagenized to Glu (R221E) and to Ala (R221A). Mutants R221E and R221A were active as wild type for ADP-ribosyltransferase and NAD glycohydrolase activity, suggesting that the arginine 221 in Yac-2 does not play a major role in enzyme activities.

• Journal of Life Science
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• v.8 no.1
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• pp.102-108
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• 1998

The nascent from of glycosylphosphatidylinositol (GPI)-anchored proteins possesses both amino and carboxy terminal hydrophobic signal sequences to direct processing in the endoplasmic reticulum (ER). Following cleavage of the amino-terminal signal peptide, the carboxy-terminal peptide is processed. Previously, mouse lymphocyte NDA: agrinine ADP-ribosyltransferase (Yac-1) was cloned and the deduced amino acid sequence of the Yac-1 transferase contained hydrophobic amino and carboxy termini, consistent with known signal sequences of GPI-anchored proteins. This tranferase was present on the surface of NMU (rat mammary adenocarcinoma) cells transfected with the wildtype cDNA and was released with phosphatidylinositol-specific phosphilpase C. Expression of the mutant protein, lacking the carboxy terminal hydrophobic sequence, resulted in the peoduction of soluble, secreted from of the transferase. This result shows that carboxy terminal sequence is important for GPI-attachment.

• Journal of Life Science
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• v.10 no.1
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• pp.20-23
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• 2000

Mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases, is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to an acceptor protein. Previously, we have identified and cloned a glycosylphosphatidylinositol-linked ADP-ribosyltransferase (Yac-1) from mouse lymphoma cells. Yac-1 enzyme contains three regions (region I,II,III) similar to those found in several bacterial toxins and vertebrate ADP-ribosyltransferases. Site-directed mutagenesis was performed to verify the role of Glu 233 in region III. Mutants E233Q, E233D and E233A were inactive for ADP-ribosyltransferase activity. Thus Glu 233 in Yac-1 is essential for enzyme activity, suggesting that Glu 233 in Glu-rich motif near the carboxy terminus plays a catalytic role in ADP-ribosyltransferase activity.