A Minimized Fc Binding Peptide from Protein A Induces Immunocyte Proliferation and Evokes Th1-Type Response in Mice 1 Pratima Sinha, 1 Jayati Sengupta, and Prasanta K. Ray 2 Immunotechnology Section, Bose Institute, Calcutta, 700 054, India Received January 28, 1999 It is now well established that PA is a potent biological response modifier, showing simultaneously antitumor, antitoxic, anticarcinogenic, antifungal, antiparasitic and immunomodulatory properties. Since PA is a for- eign protein, it is quite logical to assume that it may be cleaved into smaller peptide fragments in vivo which may be responsible for biological activities of whole PA molecule. The present study was undertaken to dissect out the structural entities of PA responsible for its bio- logical properties. Protein A (PA) of Staphylococcus au- reus has a unique property of binding with immuno- globulins. On the basis of molecular modeling and energy minimization studies a 20-mer tryptic fragment (theoretical) was predicted to retain IgG binding capac- ity which has been verified by immunoblot. This peptide sequence was selected to carry out experimental studies to show its functional mimicry of PA. We observed in the sera of 20-mer peptide treated mice that the concentra- tions of IFN, TNF and IL1 increase to a peak level by 4 h; on the other hand, there was a decrease in IL4, IL6 and IL10 concentrations at the same time (4 h). The ratio of IFN to IL4 showed Th1 type of response with the peptide as well as with that of PA. The nitric oxide con- centration in sera also increases and the peak increase was in 6 h with both the peptide and PA. Cell cycle analysis using FACS shows that 20 g dose of peptide was non-toxic to thymocytes and spleenocytes; on the other hand, it was immunoproliferative, shifting the thy- mocytes and spleenocytes from G0/G1 to S phase of the cell cycle. Further studies are in progress to evaluate other biological properties of the peptide, to evaluate if this peptide could be used as a substitute of PA to mimic at least some of its biological activities. © 1999 Academic Press Key Words: protein A; Fc binding; cytokines; Th1/Th2 pathway; immunomodulation. Protein A (PA) was discovered from the cell wall structure of Staphylococcus aureus, and utilized for a long time in the laboratory for isolation and purifica- tion of antibody molecules, specially IgG because of its Fc binding property (1). This unique Fc binding prop- erty of PA was also utilized to remove the “blocking factors” from human cancer patients (2). Along with IgG binding affinity, diverse array of biological func- tions of PA were also demonstrated, such as antitumor (3, 4), antitoxic (5), anticarcinogenic (6), immunomodu- latory (7) antifungal (8) and antiparasitic effects (9). Protein A has been demonstrated to act as B and T cell mitogen (10, 11). It also induces production of different cytokines (7), which are well known biological modula- tors regulating cellular growth and differentiation (12) on one hand, and apoptosis (13) on the other. Previous observations from our laboratory (5, 6) have demon- strated the antitoxic and anticancer properties of PA. Moreover, PA has been demonstrated to activate phase I and phase II biotransformation and detoxification enzymes, thus abrogating the toxicity of different toxic and carcinogenic chemicals (14, 15). During these stud- ies (16) we have consistently observed that PA treat- ment leads to an increase in both peripheral blood and spleenic lymphocyte population. PA induced activation of cell cycle shifting vis a vis proliferation in non- Hodgkin’s lymphoma has also been reported (17). The B-domain of protein-A, a 56 residue domain, binds the Fc portion of IgGs with dissociation constant (Kd) of about 10-50 nM (1). X-ray (18) and NMR (19) studies showed that binding contacts are presented from helix 1 (Lys7-His18) and helix 2 (Glu25-Asp36) of the B-domain (PA). It has been observed in recent years that in order to effect a biological function, large molecular structure of macromolecules may not be required always. The bio- logical function may be mediated through a portion of the whole molecule, normally called the active site or by a proteolytically degraded small peptide molecule. A particular type of contour, a globular structure, helical 1 This work was supported by DBT, Government of India, by awarding a postdoctoral fellowship to Dr. Pratima Sinha. 2 To whom correspondence should be addressed at Immunotechnol- ogy Section, Bose Institute, P-1/12 CIT Scheme VII M, Calcutta 700 054, India. Fax: 91-33-334-3886. E-mail: pkray@boseinst.ernet.in. Biochemical and Biophysical Research Communications 258, 141–147 (1999) Article ID bbrc.1999.0363, available online at http://www.idealibrary.com on 141 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.