International Journal of Poultry Science 7 (2): 125-128, 2008 ISSN 1682-8356 © Asian Network for Scientific Information, 2008 125 Peptides from the Bursa of Fabricius Associated with Suppression of Mitogen Stimulated DNA - Synthesis in Bursa of Fabricius Cells Belong to Intracellular Proteins Gary Garcia - Espinosa , Stefan Clerens , Lut Arckens , Gisela F. Erf , G. Tellez and Billy M. Hargis 1 2 2 3 3 3 Departamento de Producción Animal, Aves, Facultad de Medicina Veterinaria y Zootecnia, 1 Universidad Nacional Autónoma de México, 04510, México, D.F. Laboratory of Neuroplasticity and Neuroproteomics Katholieke Universiteit Leuven, Leuven B-3000, Belgium 2 Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA 3 Abstract: Previous reports from our laboratory have demonstrated that an reverse phase HPLC (rpHPLC) fraction obtained from extracts of the chicken bursa of Fabricius posses both in vitro anti-steroidogenic activity on avian and mammalian cells and suppression on mitogen stimulated DNA-synthesis in chicken BF cells. Utilizing YM cut-off membranes the bioactive fraction appears to be between ~3-5kDa. However, the identity of such peptide (s) remains unknown. Here, subjected those peptides for mass spectrometric (MS) nano-electrospray quadruple time-of-flight (Q-TOF) MS/MS analysis in an effort to elucidate the composition of predominant fragments. The results of these analyses indicate the presence of small fragments of the non-histone chromosomal protein high mobility group (HMG), nucleophosmin, elongation factor 1-alpha, thymosin $4 (T$4), thymosin $, stathmin and histone H1.10. These results indicate that the suppression present in the rpHPLC fraction obtained from the BF, rather than been recognized extracellular messengers, like hormones or cytokines, contains intracellular molecules. Key words: Intracellular, anti-proliferative, anti-steroidogenic and bursa of Fabricius Introduction Materials and Methods The bursa of fabricius (BF) is a primary immune organ Extraction and purification of the bioactive peptides: in avian species where early B lymphocyte proliferation The anti-steroidogenic and anti-proliferative peptides and differentiation take place (Cooper et al., 1966; Glick were extracted and purified following our previous et al., 1956; Lydyard et al., 1976). In addition, the BF has published protocol (Byrd et al., 1993). Instead of utilized been postulated to play a role in the immune-endocrine the 5kDa YM-cut off membrane we used a 10kDa FROM axis by producing a putative anti-steroidogenic factor Millipore (Bedford, MA USA). Briefly, BF were collected (Glick, 1984; Romano et al., 1981; Pedernera et al., from 7-week-old chickens immediately after death and 1985; King et al., 1985), which identity remains elusive. held at -76 C prior to extraction. Tissue (200g) was Caldwell et al., 1998 and 1999 found that a protein of homogenized with 2 parts 15% trifluroacetic acid (TFA) ~32kDa extracted and purified from the BF with (Sigma, St. Louis, MO. USA). Homogenized material was remarkable anti-steroidogenic and anti-proliferative centrifuged twice at 37,000 x g for 20 minutes at 4 C. The activity on both avian and mammalian cell culture resulting supernatant was then carefully removed, assays, which was first thought to be a hormone, has avoiding contamination with surface lipid and loaded undistinguishable relationship with the chicken histone onto 5mM TFA-equilibrated 2.5 x 10cm preparative C-18 H1 family proteins (Garcia-Espinosa et al., 2002). Mega bond elut columns (Varian, Harbor City, CA, USA). Controversially, a rpHPLC and YM cut-off membranes Cartridges were stepwise eluted (15 ml) with 0, 20, 60 purification, containing heat-labile, basic and blocked at and 80% acetonitrile (ACN) with 5 mM TFA (v:v) and both the amino and carboxyl termini peptides extracted eluent was collected in polypropylene tubes and dried by from the BF with an apparently molecular weight of ~3- vacuum centrifugation. The 60% ACN fraction, which 5kDa, shown similar suppression bioactivities on avian previously was shown to contain the bioactive peptides and mammalian cell culture assays (Byrd et al., 1993; (Byrd et al., 1993) was reconstituted in double distilled Byrd et al., 1994; Byrd et al., 1995). water and filtered (0.2μm) prior to concentration and Due the unknown identity of these peptides it is not dialysis. The solution was concentrated and dialyzed possible to establish whether or not they belong to HH1 sequentially against YM-30, YM-10 and YM-3 kDa or they are novel peptides. Therefore, our objective was centriplus units, adding milli-Q water to the sample 10 to subject those peptides to mass spectrometric (MS) times and centrifuged at 2000 x g for 45 minutes nano-electrospray quadruple time-of-flight (Q-TOF) (Amicon, Bedford, MA, USA). Resulting samples were MS/MS analysis to know their identity. dried by vacuum centrifugation, reconstitute in one ml of o o