Monoclonal antibody (MAb) productivity of human-human hybridomas is very low. Hybridomas must be enhanced their productivities to obtain enough amount of MAbs, efficiently. To boost production of MAbs of hybridoma, immunoglobulin production stimulating factor (IPSF) was screened in various cell lines. Several non-protein IPSFs such as potassium or sodium phosphate (Sato et al., 1989) and phenyl compounds (Maeda et al., 1990) were found. Suger compounds, i. e. fructose and chitosan, were also identified as IPSF.
Cellular protein IPSF was first found in the 3 M KCl extracts of human lung adenocarcinoma PC-8 cells (Shinmoto et al., 1988). IPSF-I isolated from a human lymphoblastoid cell line H0-323 was a complex protein of 410 KD (Toyoda et al., 1990). Another IPSF, IPSF-11, was purified, characterized and identified from human Burkitt's lymphoma N amalwa cells in this thesis.
IPSF-11 was obtained by purification from Namalwa cell lysate. To pre-fractionize Namalwa cell lysate by salting out, ammonium sulfate was added to the cell lysate up to a point of 50 °/o saturation. The supernatant obtained after centrifugation was then purified by hydrophobic interaction column chromatography using BUTYL TOTOPEARL 650M gel. Since the IPSF activities were broadly distributed, active fractions were grouped into three portions (A, B and C). Fraction A was further purified by gel filtration. The IPSF named I PSF-Ila was identified as a 112 KD protein composed of a 40 KD and two 36 KD subunits by SDS-PAGE analysis. The 36 KD subunit exclusively showed IPSF activity. The 40 KD subunit protein did not take part in IPSF activity. Although the role of the 40 KD protein is still unknown, there is a possibility th at it may contribute to
stabilization or transportation of the 36 KD protein in vivo. Therefore, the amino acid sequence of the 36-KD protein was analyzed for the 20 amino acid residues from the N-terminus. Surprisingly, the amino acid sequence of the protein completely coincided with that of glyceraldehyde-3-phosphate dehydrogenase (GPD) from human liver, and was highly homologous with those of GPDs derived from various origins. GPD is a one of the key enzymes in the glycolytic pathway and found in exceedingly large quantities in many cells and tissues.
Though GPDs from human muscle, rabbit muscle and B.
stearothermophilus revealed IPSF activities, there were great differences betweeng their specific IPSF activities. However, the IPSF activity of GPD was not derived from its enzymic activity, because GPD which lost enzymic activity completely retained its IPSF activity.
Immunoglobulin production of the translation-suppressed hybridomas was still stimulated by GPD. The mRNA level for immunoglobulin in the cell was not increased by the addition of sufficient amounts of GPD.
These results indicate that GPD does not accelerate transcription of mRNA from DNA to enhance immunoglobulin production. The effect of GPD on in vitro translation system was therefore examined. The addition of GPD increased immunoglobulin production in the in vitro translation system using rabbit reticulocyte lysate 50 °/o more than in the control system. Furthermore, GPD stimulated translation activity of cell-free translation system made from HB4C5 cell lysate. These
results suggest that hybridomas are stimulated translation process of protein synthesis by GPD. But, it is unclear why endogenous GPD in the hybridoma does not act as IPSF. It is commonly assumed that GPD is an inactive or low-active form that is activated during incorporation into the hybridomas. Putative modes of action of IPSF-Ila are expected that GPD activates the resting ribosome at the beginning of
the translation process. This hypothesis is supported by the fact that IPSF activity was inhibited at the higher culture temperature at which the translation process is inactivated the initiation of translation process by a marked reduction in formation of the 40 S ribosomal subunit/Met-tRNAr complex (Mizuno, 1975).
Among IPSF activities obtained by the hydrophobic interaction column chromatography, IPSF eluted by ammonium sulfate concentration 1.0-0.8 M was identified as IPSF-IIa, as described above.
Another protein possessing IPSF activity was eluted by the ammonium sulfate concentration 0. 7 M. The IPSF active fraction was purified by serial use of anion-exchange column chromatography and gel filtration.
The IPSF activity was detected in the protein fraction of which the molecular size was estimated about 45 KD. The protein was analyzed by SDS-PAGE. The most abundant protein on the gel revealed a
molecular size of 46 KD. This 46 KD protein was extracted from the gel and measured for the IPSF activity. The protein also stimulated production of immunoglobulin by hybridomas and was named IPSF-II�.
The partial amino acid sequence of IPSF- II� completely coincided with that of human enolase a-chain. Enolase is an enzyme in the glycolytic pathway, as well as GPD. Rabbit muscle enolase, which mainly contains �-chain, enhanced the production of immunoglobulin by hybridomas as well as by IPSF-II�. if used at a higher concentration, suggesting that IPSF- II� is either the a-chain itself or an isozyme.
IPSF-II� lost most of its IPSF activity when treated above 50 oc for 30 min. Its activity was completely preserved in a strong alkaline solution, though unstable in an acidic environment. Since enzymic function of the enolase loses irreversibly in such a strong alkaline condition as pH 11-13, enzymic activity of enolase does not contribute to its IPSF activity, as well as IPSF-II a . There are also some
similarities in the IPSF activities of IPSF-II a and IPSF-II�, when the modes of action of IPSF-II� are investigated and compared with IPSF
Ila. IPSF-II� was not suppressed in its IPSF activity by actinomycin D treatment of hybridomas, and it enhanced immunoglobulin synthesis of in vitro translation system using rabbit reticulocyte lysate as well as IPSF-Ila. These facts reveal that both IPSF-Ila and IPSF-II� enhance cellular protein productivity by stimulating the translation process.
Judging from all these results, there is a good possibility that the modes of action of IPSF-II� are quite similar to those of IPSF-Ila.
GPD was identified as a single-strand DNA and RNA binding protein (Perucho et al., 1980 and Ryazanov, 1985). Therefore, the IPSF activities of nucleic acid binding proteins were examined.
Lysine-rich histones such as histone Hl, H2A and H2B had IPSF activities, though arginine-rich histones did not. Since it was
expected that lysine residues contribute to the IPSF activity of these peptides, the IPSF activity of poly-L-lysine and poly-D-lysine were estimated. As expected, poly-L-lysine possessed IPSF activity. But the result that poly-D-lysine which is a biologically inactive substance showed IPSF activity was unexpected. These results indicate that IPSF activity of poly-lysine is derived from its physical or chemical features.
These substances can thus be candidates for components in the formulation of serum-free medium focused on increasing protein productivities by cultured animal cells in biotechnology.
The fact that extracellularly provided histones and IPSFs can enhance cellular protein productivities indicate that these proteins actually take part in the enhancement of the protein synthesizing process in cells. The levels of such IPSFs as GPD and enolase in cytoplasm serve to regulate cellular protein synthesis, indicating that further studies in the modulation of cellular physiology should promote
the feasibility of the use of animal cells for industrial protein productions. These studies also uncover new physiological functions of proteins such as GPD and enolase which have been believed to be enzymes in glycolytic pathway and assumed to contribute solely to energy generation. It is now clear that these enzymes have another physiological function which is to stimulate the translation of protein and be expressed irrelevant to their conventional enzymic functions.
Further studies on the uncovering of the regulating mechanisms of protein synthesis in mammalian cells will give us effective suggestions on how to make the cells fully express theie potentiality for protein production. The technology of gene engineering is expected to construct an expression system which will take all of these physiological studies in to account.
ACKNOWLEDGMENT
The author wishes to express profound gratitude and heartfelt thanks to Professor Hiraki Murakami for his many helpful suggestions, invaluable guidance and encouragement throughout this work.
The author is highly indebted to Professor Ryuzo Sasaki (Kyoto Univ.} and Professor Gunki Funatsu (Kyushu Univ.} for their helpful discussions.
The author would like to thank Dr. Sanetaka Shirahata and Mr.
Hirofumi Tachibana for their instructive discussion. The author would also like to express his thanks to Dr. Koji Yamada for his advice and encouragement.
The technical assistance were kindly supported by Mr. Kazuhiko Akiyoshi, Mr. Nobutaka Ninomiya and Miss Michiyo Nitta.
The author thanks all the staffs and their colleagues in the Laboratory of Cellular Regulation Technology, Graduate School of Genetic Resources Technology, and in the Laboratory of Food Chemistry, Department of Food Science and Technology, for their help and kindness.
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