Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin

Abstract

The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in cells of the myelomonocytic lineage and regulated by interferon alpha in a cell-specific fashion. MNDA is also a member of a family of interferon-regulated genes of unknown function. In an effort to elucidate the function of MNDA, three techniques (affinity purification, coimmunoprecipitation, and protein blot assay) were used to characterize its specific protein binding activities. Microsequence analysis showed that MNDA bound the 100 kDa nucleolin protein. The identification of nucleolin was confirmed by immunoreaction with specific antibodies. MNDA contains motifs which could account for specific binding to nucleolin. Nucleolin binds other macromolecules and exhibits features consistent with roles in signal transduction, production of ribosomes, nuclear matrix structure, and regulation of transcription. The present results indicate that the function of MNDA is most likely related to interactions with other proteins. Through these associations, MNDA could contribute cell/lineage- and differentiation-specific limits to the function of ubiquitous proteins such as nucleolin. Further analysis of MNDA protein binding could be critical to elucidating the function of MNDA and could contribute to understanding the function of the products of other members of this interferon-inducible family of genes.

The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

“…Early studies into the role of MNDA suggested an involvement in myeloid differentiation. This idea was supported by studies revealing that MNDA can bind nucleolin and nucleophosmin [14,15], both of which have been shown to be involved in the maturation and biosynthesis of ribosomes. The noted nuclear localisation, and lineage-and stage-specific expression of MNDA suggested that this PYHIN protein may play a role in regulating gene transcription.…”

MNDA dimerizes through a complex motif involving an N‐terminal basic region

“…Recombinant MNDA was prepared using the pQE30 expression system as described previously [24]. To promote 125 I-labeling primarily in the His-tag region, the purified rMNDA was first dialyzed at 4°C with three changes against 20 mM Tris-HCl pH 8.2, 500 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM PMSF to remove imidazole.…”

“…The pQE30-MNDA, described previously [24], was used to prepare His-tagged recombinant MNDA. C-terminal deletion mutations were prepared from the pQE30-MNDA construct using the Erase-a Base System (Promega).…”

“…Plaques containing the oligonucleotide-directed loop-out mutation were selected and subsequently subcloned into pQE30 for protein expression. Except where indicated, all the His-tagged MNDA mutants were induced and purified with the procedure described for the full-length MNDA [24]. Correct in-frame expression of deletion mutation constructs was verified by immunoblotting and specified site of deletion confirmed by sequencing.…”

“…The MNDA promoter [5] contains several consensus regulatory elements, including c-myb [6][7][8][9], PU.l [10][11][12][13][14][15][16][17][18], ISRE [19] and Spl [20][21][22][23], consistent with MNDA's myelomonocytic lineage-specific expression and regulation by interferon a. Analysis of MNDAassociated proteins and assessment of the effects of MNDA expression on promoter activities of different genes has led to the proposal that MNDA plays a role in transcription regulation by influencing the actions of other transcription factors [24,25]. A related mouse protein, p202, also interacts with other nuclear proteins and modulates transcription [26][27][28][29].…”