The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase.
In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.
Li
Sheng
S
Department of Biological Science, Illinois State University, Normal, IL 61790, USA.
Friesen
Jon A
JA
Fei
Hong
H
Ding
Xiang
X
Borst
David W
DW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D015139:Blotting, Southern / Q000379:methods; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007313:Insecta / Q000166:cytology; D008334:Mandible / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000201:enzymology* / Q000235:genetics; D009928:Organ Specificity; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods; D012995:Solubility; D014158:Transcription, Genetic / Q000235:genetics
D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D006903:Hydroxymethylglutaryl CoA Reductases; D010744:Phosphoric Monoester Hydrolases