A novel murine protein with no effect on iron homoeostasis is homologous with transferrin and is the putative inhibitor of carbonic anhydrase

Abstract

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.

Inequivalent Contribution of the Five Tryptophan Residues in the C-Lobe of Human Serum Transferrin to the Fluorescence Increase when Iron is Released

“…Additionally, and as reported previously (44), the single rate constant is very slow (0.46 min -1 at 295 and 0.60 min -1 at 280) requiring 500 sec to attain 5 half lives. This result contrasts with iron release from the monoferric N-lobe (Fe N hTF) that yielded three rate constants of 20.8, 4.1 and 1.1 min -1 under identical conditions and was complete in 200 sec (25). The slower rate constant for iron release from the C-lobe of Fe C hTF relative to the N-lobe in the Fe N hTF construct is attributed to differences in the second shell residues and in the hinge regions between the subdomains of each lobe.…”

Evolutionary diversification of the vertebrate transferrin multi-gene family

“…Both lobes of ICA and the C-lobe of the M subfamily have lost iron-binding capacity (Baker et al 1992; Wang et al 2007). The presence of numerous conserved clade-specific residues in the C-lobes of both of these subfamilies suggests that they have adapted to novel functions.…”

“…The following members of this family are recognized in humans: (1) serotransferrin (or serum transferrin), which is expressed in the liver and secreted into the blood serum, but also has a wider tissue expression and a variety of potential functions (Gkouvatsos et al 2012); (2) lactoferrin (Figure 1), named for its expression in milk but expressed in most biological fluids (Levay and Viljoen 1995; García-Montoya et al 2012); and (3) melanotransferrin, identified as a tumor antigen in melanoma but having a wide but much lower level of expression in normal tissues as well (Rahmanto et al 2012). Well-studied transferrin family members from non-human vertebrates include ovotransferrin, first identified from avian egg white but known to have a wider expression pattern in the domestic chicken (Giansanti et al 2012); and the inhibitor of carbonic anhydrase (ICA) reported from various non-human mammals (Wang et al 2007; Eckenroth et al 2010). …”

Evolution Reversed: The Ability To Bind Iron Restored to the N-Lobe of the Murine Inhibitor of Carbonic Anhydrase by Strategic Mutagenesis

“…As previously described, it contains the signal peptide from hTF, four amino acids from the amino terminus of hTF, a hexahistidine tag, and a Factor Xa cleavage site (19). A single mICA mutant with the S188Y mutation was first produced.…”

“…Monoferric N-His-tagged hTF constructs that bind iron only in the N-lobe (N-His Y426F/Y517F hTF-NonGly, designated Fe N hTF) or in the C-lobe (N-His Y95F/Y188F hTF NonGly, designated Fe C hTF) were similarly produced and purified. Fe N hTF served as a control for the W124R/S188Y mutant mICA construct since it binds iron only in the N-lobe (19). The homogeneity of the samples was assessed by SDS-polyacrylamide gel electrophoresis on 10% gels under reducing conditions and was visualized with Coomassie blue dye.…”