41[Noda Institute for Scientific Research GRANT]2006 Young Investigator Research Grant

Creation of Novel Catalytic Antibodies Using Yeast Cell
Surface Display System Without Mammalian Immune System
Michiko KATO-MURAI
Graduate School of Agriculture, Kyoto University

Research aims
Catalytic antibodies have the diversity of antibody molecules in the immune system in vivo as well as catalytic
functions of enzyme molecules. Catalytic antibodies havegenerally been prepared from mouse by immunization witha haptenic transition-state analog. However, the preparationof catalytic antibodies by the traditional immunization
method is time-consuming and labor-intensive at the present state. The aim of this study was to create tailor-made artificial enzymes with the desired catalytic ability in vitro
using the yeast molecular display method, which has several advantages over the immunization method, including
the ability to generate catalytic antibodies more rapidly andeasily and the ability to adapt more flexibly to highthroughput screening systems. In this study I attempted tocreate a novel antibody with serine protease-like activity byintroducing the catalytic triad composed of Ser-His-Asp
into the amino acid sequence of an antibody molecule,since catalytic antibodies with a protease activity shouldhave great application potential in medical fields.
Methods and Results
1. Introduction of catalytic triad into the light chain of
catalytic antibody 6D9Using the plasmid to display the catalytic antibody 6D91)
already constructed, the catalytic triad was designed in its
light chain (Lc). The design of the catalytic triad was performed on the basis of the sequence of anti-VIP antibody
prepared by immunization with the vasoactive intestinal
peptide (VIP). The anti-VIP antibody moderately catalyzes
the hydrolysis of VIP2) and its catalytic mechanism is similar to that of serine proteases3). Interestingly, Ser27a, His93
and Asp1 in the light chain variable region can form a serine protease-like catalytic triad4,5). From the result of alignment of amino acid sequences of light chain variable regions between the anti-VIP antibody and the catalytic antibody 6D9, it was assumed that the residues Ser27a, His93
and Asp1 in the anti-VIP antibody light chain probablyform a serine protease-like catalytic triad. In order to introduce a similar catalytic triad into the light chain of the antibody at the identical position, we carried out site-directedmutagenesis in the light chain variable region of 6D9 to replace Glu1 and Thr27a with Asp and Ser, respectively. This
double mutant was addressed as Lc (Triad) to distinguishfrom the wild-type Lc (WT). The plasmid for display of Lc(Triad) on the yeast cell surface was introduced into Saccharomyces cerevisiae BJ2168 strain, which was defectivein multiple proteases. Display of Lc (Triad) on the yeastcell surface was confirmed by observing the fluorescencearound yeast cells under a fluorescence microscope using
the immunostaining method.
2. Activity measurement of Lc (Triad) displayed on theyeast cell surface
Since the yeast molecular display method permits to display proteins on the yeast cell surface, it is possible to deal
with protein- or enzyme-displaying yeast as a protein cluster and a whole-cell biocatalyst in the case of an enzyme inparticular. Activity of Lc (Triad) displayed on the cell surface was measured without the isolation and purification ofLc (Triad).
Lc (Triad)-displaying yeast cells were cultivated at 30°C
for 24 h and then harvested by centrifugation (3000 rpm, 5min). After cells were washed twice with 50 mM Tris-HCl(pH 8.0), and resuspended for 500 m l of a cell suspension(OD60080) with the same buffer, 500 m l of 10 m g/ml
BODIPY FL casein (Molecular Probes, Eugene, OR, USA)or seven kinds of 200 m M peptide-MCA (Peptide Institute,Osaka, Japan) was added. After centrifugation of the reaction mixture, aliquot of the cell-free supernatant was transferred into a 96-well plate, and then fluorescence was measured using the Fluoroskan Ascent FL plate reader (Labsystems, Helsinki, Finland) with a filter pair of 485 nm/527 nm
for BODIPY FL casein and 390 nm/460 nm for peptideMCA, respectively. Lc (WT)-displaying yeasts were alsomeasured to compare with the activity of Lc (Triad)-displaying yeasts. When each cell suspension was reacted with
Suc-GPLGP-MCA, a substrate for collagenase-like peptidase, a remarkable difference in hydrolytic activities wasobserved between them: Lc (Triad)-displaying cells showedhigher catalytic activity than Lc (WT)-displaying cells. The
difference disappeared in the presence of the serine protease inhibitor DFP (diisopropylfluorophosphate), suggesting that the three amino acid residues, Ser27a, His93 andAsp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar manner to the anti-VIP antibody light chain. These results indicate that collagenaselike protease could be created by the introduction of thecatalytic triad into the light chain of 6D96).

42Conclusion

References

Lc (WT), the wild-type light chain of the catalytic antibody, and Lc (Triad), its mutant with Ser, His and Asp atpositions 27a, 93, and 1, respectively, were displayed on thecell surface of the protease-deficient yeast strain BJ2168.
When each cell suspension was reacted with some peptide
substrates, hydrolytic activity of Lc (Triad)-displaying cells
toward a substrate for collagenase-like peptidase was higher
than that of Lc (WT)-displaying cells. The result with theserine protease inhibitor DFP indicates that the three aminoacid residues, Ser27a, His93, and Asp1, function as a catalytic triad responsible for the proteolytic activity in a similar manner to the anti-VIP antibody light chain. Although
the molecular recognition mechanism of the Lc remains tobe investigated in detail, the introduction of additionalamino acid alternations into the light chain variable region
will enable us to further regulate the substrate specificity ofLc (Triad).

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