The structure and evolution of the murine inhibitor of carbonic anhydrase: A member of the transferrin superfamily

Abstract

The original signature of the transferrin (TF) family of proteins was the ability to bind ferric iron with high affinity in the cleft of each of two homologous lobes. However, in recent years, new family members that do not bind iron have been discovered. One new member is the inhibitor of carbonic anhydrase (ICA), which as its name indicates, binds to and strongly inhibits certain isoforms of carbonic anhydrase. Recently, mouse ICA has been expressed as a recombinant protein in a mammalian cell system. Here, we describe the 2.4 Å structure of mouse ICA from a pseudomerohedral twinned crystal. As predicted, the structure is bilobal, comprised of two a-b domains per lobe typical of the other family members. As with all but insect TFs, the structure includes the unusual reverse c-turn in each lobe. The structure is consistent with the fact that introduction of two mutations in the N-lobe of murine ICA (mICA) (W124R and S188Y) allowed it to bind iron with high affinity. Unexpectedly, both lobes of the mICA were found in the closed conformation usually associated with presence of iron in the cleft, and making the structure most similar to diferric pig TF. Two new ICA family members (guinea pig and horse) were identified from genomic sequences and used in evolutionary comparisons. Additionally, a comparison of selection pressure (dN/dS) on functional residues reveals some interesting insights into the evolution of the TF family including that the N-lobe of lactoferrin may be in the process of eliminating its iron binding function.

Prenatal Endotoxin Exposure Induces Fetal and Neonatal Renal Inflammation via Innate and Th1 Immune Activation in Preterm Pigs

“…As HIF1A was also up-regulated by IA LPS in the current study, we speculate that LRG1-mediated hypoxia via HIF-1α activation also contributes to kidney injury and that LRG1 may be a promising diagnostic and therapeutic target of prenatal inflammation and organ responses including neonatal renal inflammation. On the other hand, the ICA is a CA inhibiting protein and pH (acid-base balance) regulating enzyme in multiple cells and tissues (77)(78)(79), although in humans it may only be a pseudogene (80). Instead, we found elevated expression of renal carbonic anhydrase II (CA2) in LPS pigs at birth and speculated that the imbalance between ICA and CA2 may enhance bicarbonate generation and low tissue pH, contributing to inflammation-related kidney dysfunction.…”

High-density chemical cross-linking for modeling protein interactions

“…The murine inhibitor of CA (mICA) was shown to be a nanomolar inhibitor of several isoforms, including the ubiquitous CAII enzyme (35). The structures of CAII in different species (36,37) have been well studied, and the structure of mICA was recently solved (38); however, the structure of the enzyme-inhibitor complex remains elusive (35). Identifying Human as well as bovine CAII with both cross-linkers showed strong Coomassie-stained bands corresponding to a 1:1 complex (Fig.…”

Evolutionary diversification of the vertebrate transferrin multi-gene family

“…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). …”

“…Similar three-dimensional structures have been reported for serotransferrins and lactoferrins of a variety of mammalian species and for ovotransferrins of two bird species (Mizutani et al 2012). Mammalian ICA does not bind iron, but has a three-dimensional structure resembling that seen in other TFs when they are binding iron (Eckenroth et al 2010). A three-dimensional structure for melanotransferrin has not yet been determined, but melanotransferrin is known to differ from the other vertebrate transferrins in that it attaches to the cell membrane though a glycosyl phostphatidylinositol linkage involving a hydrophobic domain located at the C-terminus of the C-lobe (Alemany et al 1993).…”