Human histamine N-methyltransferase pharmacogenetics: cloning and expression of kidney cDNA

B Girard, DM Otterness, TC Wood, R Honchel, ED Wieben and RM Weinshilboum

Department of Pharmacology, Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905.

Histamine N-methyltransferase (HNMT) catalyzes the NT-methylation of histamine. The level of HNMT activity in human red blood cells is controlled by a common genetic polymorphism. We set out to clone and express a cDNA for HNMT from human tissue as a first step toward a determination of the molecular basis for this genetic polymorphism. The cloning strategy was based on possible sequence homology between rat and human kidney HNMT. Human kidney cDNA libraries were screened with the 885-nucleotide open reading frame of rat kidney HNMT cDNA. A 1.4- kilobase cDNA clone was isolated that contained two potential translation initiation codons, both in the same reading frame. The longest open reading frame of the human kidney cDNA clone contained 876 nucleotides and encoded a protein 292 amino acids in length. The amino acid sequence of this protein was 84% identical to that of rat kidney HNMT. The human kidney cDNA clone was transcribed in vitro and translated in a rabbit reticulocyte lystate system to yield a protein with an apparent molecular mass of 33 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human kidney cDNA was also subcloned into the eukaryotic expression vector p91023(B). Partially purified HNMT isolated from the cytosol of GOS-1 cells transfected with this expression construct had biochemical properties similar to those of human kidney HNMT. Human renal cortical HNMT, partially purified human renal cortical HNMT, and partially purified transfected COS-1 cell HNMT had Km values for histamine and S- adenosyl-L-methionine, the two cosubstrates for the enzyme reaction, of 20, 13, and 14 microM and 2.0, 3.0, and 6.2 microM, respectively. IC50 values for the HNMT inhibitor amodiaquine were 0.50, 0.48, and 0.40 microM, respectively, for enzyme from these same three sources. Northern blot analyses performed with poly(A)+ RNA from a series of human tissues including kidney demonstrated three transcripts, approximately 1.3, 3.8, and 4.0 kilobases in length. Cloning of a cDNA for HNMT may now make it possible to determine the molecular basis for the HNMT genetic polymorphism in humans.

Volume 45, Issue 3, pp. 461-468, 03/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics

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