title: Neutrophil elastase-processing defect in cyclic hematopoietic dogs.
abstract: OBJECTIVE
  Canine cyclic hematopoiesis (CH), a model of human cyclic neutropenia and severe congenital neutropenia, is characterized by a periodic reduced neutrophil count and decreased neutrophil elastase (NE) enzymatic activity. Canine CH is caused by a mutation of AP3B1 encoding the beta3A subunit of adaptor protein complex-3 (AP-3). It has been proposed that trafficking of elastase is affected by AP-3. The aim of this study was to study intracellular sorting/trafficking of NE in CH dogs using antibodies specific to canine NE.
  
  
  METHODS
  Polyclonal and monoclonal antibodies were generated to immunogenic epitopes in the middle (aa85-98) and C-terminal (aa269-282) regions of NE. The antibodies to canine NE were characterized by Western immunoblotting and immunocytochemistry.
  
  
  RESULTS
  Antibody ELA85 (antibody to canine NE aa 85-98) specifically recognized mature 28-kD NE. Immunocytochemical analysis using ELA85 and an antibody to myeloperoxidase demonstrated colocalizaton of NE and myeloperoxidase in primary granules of normal dogs. Antibody ELA269 (antibody to canine NE aa 269-282) reacted exclusively with the 33-kD NE presumptive precursor form. Immunocytochemical analysis demonstrated that the NE precursor was not colocalized with myeloperoxidase in the primary granules of normal or CH dogs. Western immunoblotting using these antibodies demonstrated that CH dogs contained reduced mature NE, but accumulated a large amount of the NE precursor protein that was not enzymatically active.
  
  
  CONCLUSIONS
  Antibodies ELA85 and ELA269 were found to be useful reagents for studying the biosynthesis, processing, and trafficking of NE during normal myelopoiesis. Neutrophils from CH dogs accumulated large amounts of higher molecular weight elastase precursors compared to normal dogs.
authors: Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, Ala., USA.
authors: Ronghua
authors: R
authors: Meng
authors: Roger
authors: R
authors: Bridgman
authors: Maria
authors: M
authors: Toivio-Kinnucan
authors: Glenn P
authors: GP
authors: Niemeyer
authors: William
authors: W
authors: Vernau
authors: Tommy
authors: T
authors: Hock
authors: Clinton D
authors: CD
authors: Lothrop
mesh_terms: D033963:Adaptor Protein Complex 3 / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D003594:Cytoplasmic Granules / Q000201:enzymology; D004283:Dog Diseases / Q000201:enzymology* / Q000235:genetics; D004285:Dogs; D004792:Enzyme Precursors / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D006410:Hematopoiesis; D007150:Immunohistochemistry; D019272:Leukocyte Elastase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D009154:Mutation; D009503:Neutropenia / Q000235:genetics / Q000662:veterinary*; D009504:Neutrophils / Q000201:enzymology / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D009195:Peroxidase / Q000032:analysis / Q000276:immunology
chemical_list: D033963:Adaptor Protein Complex 3; D000906:Antibodies; D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D010446:Peptide Fragments; D009195:Peroxidase; D019272:Leukocyte Elastase