| Autor: |
Wang F; Division of Hematology/Oncology, Children's Hospital Boston, and Harvard Medical School, Boston, MA 02115, USA., Lothrop AP, James NG, Griffiths TA, Lambert LA, Leverence R, Kaltashov IA, Andrews NC, MacGillivray RT, Mason AB |
| Abstrakt: |
In a search for genes that modify iron homoeostasis, a gene (1300017J02Rik) was located immediately upstream of the murine TF (transferrin) gene. However, expression of the 1300017J02Rik gene product was not responsive to a number of modulators of iron metabolism. Specifically, expression was not altered in mouse models of iron disorders including mice with deficiencies in the haemochromatosis protein Hfe, the recombination-activating protein, Rag, beta2-microglobulin, TF, ceruloplasmin or Hb, or in mice with microcytic anaemia. Additionally, neither lipopolysaccharide nor hypoxia treatment resulted in any significant changes in the 1300017J02Rik expression level. The genomic DNA sequence suggested that the 1300017J02Rik gene product might be a protein equivalent to the pICA {porcine ICA [inhibitor of CA (carbonic anhydrase)]}. The coding region for the murine 1300017J02Rik gene was placed into the pNUT expression vector. Transformed BHK cells (baby-hamster kidney cells) were transfected with this plasmid, resulting in secretion of recombinant mICA (murine ICA) into the tissue culture medium. Following purification to homogeneity, the yield of mICA from the BHK cells was found to be considerably greater (at least 4-fold) than the yield of pICA from a previously reported Pichia pastoris (yeast) expression system. MS showed that the recombinant mICA was a glycoprotein that associated with CA in a 1:1 stoichiometry. Despite its high sequence similarity to TF, titration experiments showed that mICA was unable to bind iron specifically. Although enzymatic assays revealed that mICA was able to inhibit CA, it is unclear if this is its sole or even its major function since, to date, humans and other primates appear to lack functional ICA. Lastly, we note that this member of the TF superfamily is a relatively recent addition resulting from a tandem duplication event. |
