ABSTRACT
Mass spectrometric characterization of the surfactant protein A (SP-A) receptor 210 (SP-R210) led to the identification of myosin (Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated against the unique C-terminal tail of MyoXVIIIA revealed that MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow, spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells stably transfected with either MyoXVIIIA or MyoIIA indicated that SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA domains expressed in bacteria mapped the epitopes of previously derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the binding of SP-A to macrophages. Together, these findings indicate that MyoXVIIIA constitutes a novel receptor for SP-A.
Subject(s)
Myosins/chemistry , Receptors, Cell Surface/chemistry , Amino Acid Sequence , Animals , Bacteria/metabolism , Base Sequence , Blotting, Northern , Blotting, Western , COS Cells , Cell Membrane/metabolism , Cells, Cultured , Chlorocebus aethiops , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Epitopes/chemistry , Flow Cytometry , Humans , Immunoglobulin G/chemistry , Immunoprecipitation , Macrophages/metabolism , Mass Spectrometry , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Myosins/physiology , Nonmuscle Myosin Type IIA/chemistry , Peptides/chemistry , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Pulmonary Surfactant-Associated Protein A/chemistry , Rats , Recombinant Proteins/chemistry , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Distribution , Transfection , U937 CellsABSTRACT
An IS3-family insertion element, IS999, was identified in the opportunistic pathogen Mycobacterium avium. The 1347 bp element has 29 bp inverted repeats and two overlapping open reading frames coding for putative transposases. It was detected in the genomes of ten of 12 M. avium isolates examined. Copy numbers ranged from four to 16. IS999 is less stable than IS1245, the most commonly-used marker for typing M. avium isolates. Among 60 colonies picked from a single patient isolate, there were two distinct IS1245 restriction fragment length polymorphism banding patterns compared to eight distinct IS999 patterns (five in one IS1245 group and three in the other). In view of its instability, we asked whether transposition of IS999 might have phenotypic consequences. Nucleotide sequence analysis of insertion sites in four isolates revealed 16 putative structural genes that were variably disrupted by IS999. Insertions into hdhA, a gene that codes for a putative short chain alcohol dehydrogenase, were distributed non-randomly between colony type variants, consistent with phenotypic consequences that exert selective pressure. These observations illustrate the genetic heterogeneity that can exist within populations of M. avium that appear to be homogeneous by IS1245 analysis. IS999 may be a useful marker for tracking, at the sub-strain level, the rapid genetic drift that M. avium isolates undergo in nature and in the laboratory.
