A novel function of enolase from rabbit muscle; an immunoglobulin production stimulating factor

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Abstract

Rabbit muscle enolase stimulates immunoglobulin production by a human hybridoma line, HB4C5 cells under serum-free condition. IgM productivity of HB4C5 cells was enhanced more than 20-fold by this enzyme at 220 μg/ml. Human peripheral blood lymphocytes were also facilitated their IgM and IgG productivity in the serum-free medium. However, baker's yeast enolase was ineffective to accelerate immunoglobulin production by HB4C5 cells, in spite of the same specific enzymatic activity as rabbit muscle enolase. There were differences in sensitivities against heat treatment and trypsin digestion between IPSF and enzymatic activities of enolase. These results imply that the immunoglobulin production stimulating effect of rabbit muscle enolase is irrelevant to its enzymatic function and reaction products. This fact also means that this enzyme has another function other than enzymatic one in glycolysis. Rabbit muscle enolase enhanced IgM production of transcription-suppressed HB4C5 cells treated with actinomycin D. Cycloheximide treatment of HB4C5 cells was useless to inhibit the expression of immunoglobulin production stimulating activity. However, inhibition of post-transcriptional process by monensin invalidated the activity of enolase. These findings suggest that enolase from rabbit muscle accelerates the steps between translation and post-translational processes to enhance immunoglobulin productivity. In addition, laser confocal microscopic analysis revealed that enolase from rabbit muscle was subsequently incorporated by HB4C5 cells.

Introduction

The lower productivity of animal cells in vitro is due to the decrease in protein synthesis activity. The protein synthesis activity of animal cells in vitro is much lower than that in vivo. However, animal cells have the latent ability to produce a large amount of proteins. Indeed, a plasma cell in lymph nodes secrets 2000 antibody molecules/s; i.e. 1.7×108 mol/day, corresponding to 43 pg IgG/cell/day in vivo [1]. However, this enormous rate of protein synthesis by cell lines was not recognized in conventional culture conditions in vitro. At this point of view, we have paid attention to stimulation of immunoglobulin (Ig) production by hybridomas and lymphocytes in vitro, especially serum-free conditions.

Several factors termed Ig production stimulating factor (IPSF) were discovered in cell extracts and culture media. The first IPSF, IPSF-I, was purified as a 410 kDa macro-protein from culture medium of HO-323 cells derived from human lymphoblastoid cell [2]. IPSF-I stimulates IgM productivities of human and mouse hybridomas under serum-free conditions. Cell lysate of human Burkitt's lymphoma line, Namalwa cells, also contained a factor which enhances Ig production by wide variety of human and mouse hybridomas. The factor was named as IPSF-II, and purified. As the result of purification, two active substances were found in Namalwa cell lysate, i.e. IPSF-IIα and IPSF-IIβ. IPSF-IIα was completely purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12), a key enzyme in the glycolytic pathway [3]. GAPDH stimulated IgM production by various human and mouse hybridomas more than 10-fold in serum-free medium. Moreover, Ig production by human peripheral blood lymphocytes (PBL) was also accelerated by GAPDH in vitro. Ig production stimulating activity of this glycolytic pathway enzyme was derived from neither its enzymatic activity nor reaction products, such as 1,3-bisphosphoglycerate. Transcription-suppressed hybridomas treated with actinomycin D (Act D) were enhanced their Ig production as well as intact hybridomas by GAPDH. However, this enzyme did not facilitate Ig production by translation-suppressed hybridomas treated with cycloheximide. These facts suggest that GAPDH has another biological function, that is activation of protein synthesis on the post-transcriptional process [4].

Namalwa cell lysate contained another IPSF-II, that is IPSF-IIβ. IPSF-IIβ was also purified and identified as enolase (EC 4.2.1.11) on the basis of partial amino acid sequence and its enzymatic activity [5]. Enolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Enolase from a number of origins have been purified and the gene structures were determined 6, 7, 8, 9, 10. Mammals have three genes for enolase; their protein structures are the α-, β-, and γ-subunits. Most enolase molecules form dimers. Both homo- and hetero-dimers are formed with five of the six possible dimers reported to exist in vivo 11, 12, 13. Enolase present in mature rabbit muscle is ββ-dimer, and the isozyme has been purified and studied, extensively 14, 15, 16. However, there is no reports mentioned about immune functions and protein synthesis activating features of this isozymes. Then, we report here that the effect of enolase from rabbit muscle on Ig production, and its mode of actions as IPSF.

Section snippets

Materials

Enolase from rabbit muscle (r-enolase) was purchased from Boehringer Mannheim (Germany). Enolase from baker's yeast (y-enolase) was obtained from Sigma (USA). These enzymes were dialyzed against 10 mM sodium phosphate buffer (pH 7.4) and sterilized by filtration before use. Act D, cycloheximide and monensin were obtained from Wako Pure Chemistry (Japan). Act D and monensin were dissolved in ethanol, cycloheximide was dissolved in water. Trypsin was obtained from Difco (USA), and soybean trypsin

Effect of r-enolase on Ig production by HB4C5 cells

Human hybridoma HB4C5 cells were cultured in ITES–ERDF supplemented with r-enolase at various concentrations to investigate the effect of this enzyme on enhancement of IgM production. After 6 h-cultivation, the amount of IgM in the culture medium was measured by ELISA. HB4C5 cells produced 5.3±0.4 ng IgM/105 cells for 6 h in ITES–ERDF medium. When r-enolase was added at 220 μg/ml, the amount of IgM in culture medium reached 108.1±7.2 ng/105 cells. This result means that r-enolase stimulates IgM

Discussion

We previously reported about the purification of an Ig production stimulating factor derived from a human Burkitt's lymphoma line, Namalwa cells [23]. Namalwa cell extracts contained two IPSF active substances, namely IPSF-IIα and -IIβ. IPSF-IIα was completely purified as a 112 kDa protein that has a subunit structure composed of a 40 kDa and two homologous 36 kDa polypeptides [24]. The 36 kDa subunit exclusively retained IPSF activity, and the sequence of 20 amino acids from N-terminus was highly

References (27)

  • T. Sugahara et al.

    FEBS Lett.

    (1995)
  • C.C. Rider et al.

    Biochim. Biophys. Acta

    (1974)
  • A. Holt et al.

    J. Biol. Chem.

    (1961)
  • M.J. Holland et al.

    J. Biol. Chem.

    (1981)
  • H. Murakami

    Cytotechnology

    (1990)
  • K. Toyoda et al.

    Cytotechnology

    (1991)
  • T. Sugahara et al.

    Cytotechnology

    (1991)
  • T. Sugahara et al.

    Cytotechnology

    (1992)
  • K. Sakimura et al.

    Nucleic Acids Res.

    (1985)
  • A. Giallongo et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1986)
  • G.A. Russell et al.

    Biochem. J.

    (1986)
  • S.M. McAleese et al.

    Eur. J. Biochem.

    (1988)
  • L. Cali et al.

    Nucleic Acids Res.

    (1990)
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