Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is expressed in melanoma cells

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Abstract

Sperm-specific glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is normally expressed only in sperms, but not in somatic tissues. Analysis of the expression of GAPDS mRNA in different cancer cell lines shows that the content of GAPDS mRNA is enhanced in some lines of melanoma cells. The purpose of the study was to assay melanoma cells for the expression of protein GAPDS. Three different lines of melanoma cells were investigated. By data of Western blotting, all investigated cells contain a 37-kDa fragment of GAPDS polypeptide chain, which corresponds to the enzyme GAPDS lacking N-terminal amino acid sequence that attaches the enzyme to the cytoskeleton of the sperm flagellum. The results suggest that GAPDS is expressed in melanoma cells without N-terminal domain. The immunoprecipitation of proteins from melanoma cell extracts using rabbit polyclonal antibodies against native GAPDS allowed isolation of complexes containing 37-kDa subunit of GAPDS and full-length subunit of somatic glyceraldehyde-3-phosphate dehydrogenase (GAPD). The results indicate that melanoma cells express both isoenzymes, which results in the formation of heterotetrameric complexes. Immunocytochemical staining of melanoma cells revealed native GAPDS in the cytoplasm. It is assumed that the expression of GAPDS in melanoma cells may facilitate glycolysis and prevent the induction of apoptosis.

Highlights

► Melanoma cells were assayed for expression of sperm-specific glycolytic enzyme GAPDS. ► GAPDS fragment lacking N-terminal amino acid sequence is revealed in melanoma cells. ► The revealed GAPDS species form hybrid tetramers with somatic isoenzyme GAPD. ► Immunochemical staining of melanoma cells revealed native GAPDS in the cytoplasm.

Introduction

Genes expressed both in normal testes and in malignant tumors (Cancer/testes associated (СТА) genes) form a gene group that is being intensively investigated since it is important for the understanding of the mechanisms of the tumor genesis and for the search of new methods of diagnostics and treatment of the cancer. Nearly 100 CTA genes have been already identified, and their number is constantly growing. Nevertheless, a question that is constantly raised is whether the expression of testes-specific genes in cancer tumors is a random process accounted for the instability of the genome in cancer cells, or it is a specific feature of the growing tumor that is necessary for its development.

One of the specific features of cancer cells is the change in their metabolism. In normal cells, the functioning of the mitochondrial system of oxidative phosphorylation in the presence of oxygen results in the inhibition of glycolysis (Pasteur effect), but this effect is absent in cancer cells. Moreover, cancer cells are characterized by not only high intensity of glycolysis (anaerobic pathway of metabolism), but also by inhibition of respiration (Crabtree effect [1]). There is no unambiguous interpretation of these effects and their mechanisms, but it is assumed that the compartmentalization of glycolytic enzymes and interactions between them, as well as specific features of catalytic and regulatory mechanisms must be of importance for the regulation of the balance between glycolysis and oxidative phosphorylation.

It may be assumed that the expression of sperm-specific glycolytic enzymes in cancer cells could change the regulation of glycolysis and the coupled metabolic pathways. The expression of one of glycolytic enzymes, sperm-specific lactate dehydrogenase C was revealed in different human cancer tumors, and this enzyme was suggested to be responsible for the constitutive activation of the anaerobic pathway in cancer cells [2].

It should be noted that sperm-specific isoforms of glycolytic enzymes significantly differ from corresponding somatic isoenzymes in a number of catalytic and regulatory parameters, including stability and compartmentalization. The main function of these enzymes is to supply energy for the contractive elements of the principal part of the sperm flagellum providing progressive movement of the sperm (mitochondria were shown to provide only 20% of the energy [3]). Some glycolytic enzymes are firmly attached to the cytoskeleton of the flagellum (so called fibrous sheath [4]).

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) significantly differs from the somatic isoenzyme GAPD. GAPDS possesses an additional N-terminal sequence (72 amino acid residues in human) providing the attachment of the enzyme to the fibrous sheath of the sperm flagellum [5], [6] and exhibits an enhanced stability [7] compared to the somatic enzyme GAPD. Besides, the amino acid sequence of GAPDS lacks the motifs that are responsible for the translocation of the somatic enzyme GAPD through the nuclear membrane [8]. Thus, GAPDS cannot be involved in the apoptosis, the process controlled by the somatic GAPD [9], [10]. We assumed that GAPDS could be expressed in cancer cells, and the emergence of the protein with properties that differed from those of the somatic isoenzyme could significantly affect their metabolism and proliferation.

We analyzed information on the expression of GAPDS mRNA in 15322 samples that is available in the ArrayExpress Database (www.ebi.ac.uk/arrayexpress, accession numbers E-TABM-185, E-GEOD-2109, E-MTAB-37, E-MTAB-62, E-GEOD-7127, E-GEOD-10843 and E-GEOD-7307). By these data, the expression of GAPDS mRNA in most cancer cell lines, as well as in somatic tissues is virtually absent. At the same time, in some investigated melanoma cell lines, the content of GAPDS mRNA is close to its content in the testes. Consequently, the sperm-specific enzyme GAPDS could be found in melanoma cells.

In the present work, using different methods, we identified sperm-specific glyceraldehyde-3-phosphate dehydrogenase in several lines of human melanoma cells. Thus, subsequent investigation of this protein could be useful for the understanding of the role of GAPDS in the altered metabolism and proliferation of cancer cells.

Section snippets

Materials and methods

Melanoma cell lines MelIL, MelKor, and MelP were obtained previously during the collaborative work with Blokhin Cancer Research Center of Russian Academy of Medical Sciences and Petrov Research Institute of Oncology [11]. Primary cultures were isolated from metastatic tumors of patients suffering from melanoma. All cell lines were cultivated in a RPMI-1640 medium (HyClone, USA) with the addition of 10% fetal bovine serum (HyClone), 2 mM l-glutamine (HyClone), 100 U/ml penicillin (Sintez, Russia),

Results and discussion

Analysis of the expression of GAPDS mRNA in different cancer tissues showed an increased level of GAPDS mRNA in some melanoma cell lines (ArrayExpress database, www.ebi.ac.uk/arrayexpress, accession numbers E-MTAB-37, E-MTAB-62, E-GEOD-10843 and E-GEOD-7127). This suggested that the sperm-specific enzyme GAPDS could be revealed in melanoma cells.

On the first stage of our investigations, we assayed three different lines of melanoma cells for expression of GAPDS by Western blotting of the

Acknowledgments

This work was supported by the Russian Foundation of Basic Research (11-08-00663-а and 12-04-91330 NNiO_a), and DFG International Research Training Group “Regulation and Evolution of Cellular Systems” (GRK 1563).

References (18)

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