Elevation of gamma-glutamyltransferase activity in 293 HEK cells constitutively expressing antisense glutaminase mRNA.
Previous studies have shown that the use of dynamic nutrient feeding to maintain glutamine at low levels in fed-batch cultures reduced the overflow of glutamine metabolism. This strategy resulted in the shift of metabolism towards an energetically more efficient state signified by reduced lactate and ammonia production and thus achieving a higher cell density for enhanced productivity. In an effort to mimic the metabolic changes effected by this fed-batch strategy at the molecular level, 293 HEK cells were engineered via stable transfection with an antisense fragment of the rat phosphate-dependent glutaminase (PDG) gene. PDG is localized in the mitochondria and catalyzes the deamination of glutamine to glutamate with the release of ammonia. Stable single cell clones were isolated from the transfected populations. Characterization of these transfectants revealed indications of an altered glutamine metabolism affected by the antisense strategy. Contrary to our expectations, glutamine consumption and ammonia production in the antisense cells did not deviate significantly from that of untransfected cells. Glutamate was also observed to accumulate to high level extracellularly, as opposed to a consumption pattern normally observed in non-transfected cells. Subsequent analyses show that gamma-glutamyltransferase (gamma-GT) may be a significant pathway that resulted in the formation of glutamate and ammonia from glutamine catabolism extracellularly. gamma-GT has been widely investigated in renal glutamine metabolism, but has rarely been implicated in cultured cell metabolism. This study highlights the importance of this alternative glutamine metabolism pathway in cell culture.
Wong
Kathy T K
KT
Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore. kathy_wong@bti.a-star.edu.sg
Lee
Yih Yean
YY
Brusic
Vesna
V
Tan
Janice
J
Yap
Miranda G S
MG
Nissom
Peter Morin
PM
D000641:Ammonia / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D020868:Gene Silencing; D018698:Glutamic Acid / Q000378:metabolism; D005972:Glutaminase / Q000235:genetics* / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000378:metabolism*
D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D000641:Ammonia; D005723:gamma-Glutamyltransferase; D005972:Glutaminase