Impact of two novel mutations on the structure and function of human myeloperoxidase.
The heme protein myeloperoxidase (MPO) contributes critically to O(2)-dependent neutrophil antimicrobial activity. Two Japanese adults were identified with inherited MPO deficiency because of mutations at Arg-499 or Gly-501, conserved residues near the proximal histidine in the heme pocket. Because of the proximity of these residues to a critical histidine in the heme pocket, we examined the biosynthesis, function, and spectral properties of the peroxidase stably expressed in human embryonic kidney cells. Biosynthesis of normal MPO by human embryonic kidney cells faithfully mirrored events previously identified in cells expressing endogenous MPO. Mutant apopro-MPO was 90 kDa and interacted normally with the molecular chaperones ERp57, calreticulin, and calnexin in the endoplasmic reticulum. However, mutant precursors were not proteolytically processed into subunits of MPO, although secretion of the unprocessed precursors occurred normally. Although delta-[(14)C]aminolevulinic acid incorporation demonstrated formation of pro-MPO in both mutants, neither protein was enzymatically active. The Soret band for each mutant was shifted from the normal 430 to approximately 412 nm, confirming that heme was incorporated but suggesting that the number of covalent bonds or other structural aspects of the heme pocket were disrupted by the mutations. These studies demonstrate that despite heme incorporation, mutations in the heme environs compromised the oxidizing potential of MPO.
Goedken
Melissa
M
Inflammation Program, Department of Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52241, USA.
McCormick
Sally
S
Leidal
Kevin G
KG
Suzuki
Kazuo
K
Kameoka
Yosuke
Y
Astern
Joshua M
JM
Huang
Meilan
M
Cherkasov
Artem
A
Nauseef
William M
WM
D000622:Aminolevulinic Acid / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D037281:Calnexin / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D007668:Kidney / Q000196:embryology; D008958:Models, Molecular; D020125:Mutation, Missense; D010100:Oxygen / Q000737:chemistry; D009195:Peroxidase / Q000235:genetics* / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism
D037282:Calreticulin; D037281:Calnexin; D006418:Heme; D000622:Aminolevulinic Acid; D001120:Arginine; D010544:Peroxidases; D009195:Peroxidase; D010100:Oxygen; D005998:Glycine