Research article

Characterization of human brain nicotinamide 5′-mononucleotide adenylyltransferase-2 and expression in human pancreas

Joel A. YALOWITZ, Suhong XIAO, Mangatt P. BIJU, Aśok C. ANTONY, Oscar W. CUMMINGS, Mark A. DEEG, Hiremagalur N. JAYARAM

Abstract

NMNAT (nicotinamide 5´-mononucleotide adenylyltransferase; EC 2.7.7.1) catalyses the transfer of the adenylyl group from ATP to NMN to form NAD. We have cloned a novel human NMNAT cDNA, designated hNMNAT-2, from human brain. The cDNA contains a 924 bp open reading frame that encodes a 307 amino acid peptide that was expressed as a histidine-patch-containing thioredoxin fusion protein. Expressed hNMNAT-2 shared only 35% amino acid sequence homology with the human NMNAT enzyme (hNMNAT-1), but possessed enzymic activity comparable with hNMNAT-1. Using human genomic databases, hNMNAT-2 was localized to chromosome 1q25 within a 171 kb gene, whereas hNMNAT-1 is on chromosome 1p32–35. Northern blot analysis revealed highly restricted expression of hNMNAT-2 to brain, heart and muscle tissues, which contrasts with the wide tissue expression of hNMNAT-1; different regions of the brain exhibited differential expression of hNMNAT-2. Substitution mutations of either of two invariant residues, His-24 or Trp-92, abolished enzyme activity. Anti-peptide antibody to a unique epitope within hNMNAT-2 was produced, and immunohistochemical analysis of sections of normal adult human pancreas revealed that hNMNAT-2 protein was markedly expressed in the islets of Langerhans. However, the pancreatic exocrine cells exhibited weak expression of hNMNAT-2 protein. Sections of pancreas from insulinoma patients showed strong expression of hNMNAT-2 protein in the insulin-producing tumour cells, whereas acinar cells exhibited relatively low expression of hNMNAT-2 protein. These data suggest that the unique tissue-expression patterns of hNMNAT-2 reflect distinct functions for the isoforms in the regulation of NAD metabolism.

  • enzyme kinetics
  • human NMN adenylyltransferase
  • immunohistochemistry
  • NAD metabolism
  • protein expression
  • protein purification

Footnotes

  • Abbreviations used: EST, expressed sequence tag; NMNAT, nicotinamide mononucleotide adenylyltransferase; hNMNAT, human NMNAT; RT-PCR, reverse transcriptase–PCR; TAD, thiazole-4-carboxamide adenine dinucleotide; IDDM, insulin-dependent diabetes mellitus.