Human liver-specific very-long-chain acyl-coenzyme A synthetase: cDNA cloning and characterization of a second enzymatically active protein.
Activation of fatty acids, catalyzed by acyl-coenzyme A (acyl-CoA) synthetases, is required for their subsequent metabolism. Peroxisomes and microsomes contain very-long-chain acyl-CoA synthetases (VLCSs) capable of activating fatty acids with a chain length of 22 or more carbons. Decreased peroxisomal VLCS activity is, in part, responsible for the biochemical pathology in X-linked adrenoleukodystrophy (X-ALD), illustrating the importance of VLCSs in cellular fatty acid homeostasis. We previously cloned two human genes encoding proteins homologous to rat peroxisomal VLCS; one (hVLCS) is the human ortholog to the rat VLCS gene and another (hVLCS-H1) encodes a related heart-specific protein. Here, we report the cloning of a third gene (hVLCS-H2) and characterization of its protein product. The hVLCS-H2 gene is located on human chromosome 19 and encodes a 690-amino-acid protein. The amino acid sequence of hVLCS-H2 is 44-45% identical and 67-69% similar to those of both hVLCS and hVLCS-H1. COS-1 cells transiently overexpressing hVLCS-H2 activated the very-long-chain fatty acid lignocerate (C24:0) at a rate >1.5-fold higher than that of nontransfected cells (P < 0.002). The hVLCS-H2-dependent activation of long- and branched-chain fatty acids following transient transfection was less striking. However, hVLCS-H2-dependent acyl-CoA synthetase activity with long- and very-long-chain fatty acid substrates was detected in COS-1 cells stably expressing hVLCS-H2. For all substrates tested (C18:0, C20:0, C24:0, C26:0), the hVLCS-H2 catalyzed activity was significantly increased (P < 0.01 to P < 0.0001). By both Northern analysis and reverse transcription polymerase chain reaction, hVLCS-H2 is expressed primarily in liver. Indirect immunofluorescence of COS-1 cells or human hepatoma-derived HepG2 cells expressing epitope-tagged hVLCS-H2 revealed that the protein was associated with the endoplasmic reticulum but not with peroxisomes. Thus, the primary role of hVLCS-H2 is likely to be in fatty acid elongation or complex lipid synthesis rather than in degradation.
Steinberg
S J
SJ
Kennedy Krieger Institute and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Wang
S J
SJ
McGuinness
M C
MC
Watkins
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002874:Chromosome Mapping; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000737:chemistry; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D018076:DNA, Complementary; D007527:Isoenzymes; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase