Heme Oxygenase: Isoforms

The heme oxygenases are key enzymes in the conversion of heme to biliverdin IXα, CO, and ferrous iron (Fe²⁺) ¹, constituting the heme oxygenase (HO) family of heme-degrading enzymes which is also known as HSP32 family. The human HO family comprises at least two functional protein-coding genes, while in rats two protein-coding genes and one pseudogene have been described. The corresponding gene products differ from each other by expression level, subcellular location and amino acid constitution (see also section Family Members and Table 2). In mammals, two isoforms are expressed: HO-1 and HO-2 (Figure 3). While HO-2 is constitutively expressed under basic requirements in the majority of human tissues, HO-1 represents the inducible HO variant whose expression is highly up-regulated upon exposure to different kinds of stress ^2-4. Enzymatic analyses disclosed that v_max of HO-2 and the K_m for heme are approximately one-tenth or three times that of HO-1, respectively ^{65, 66}. The coding sequence of the expressed HO-1 protein consists of 288 amino acid residues yielding a molecular mass of 32.8 kDa. A novel splice variant of human HO-1 with a molecular mass of 14 kDa has most recently been identified by Bian and colleagues.  This 14 kDa variant could be detected in the cytoplasm of malignant cells where it promotes cell proliferation and increases the relative length of telomeres in vivo and in vitro ⁶. This 14 kDa HO-1 lacks aa 49 – 212 of full-length HO-1 and is unable to bind heme ⁶. Unlike HO-1, HO-2 bears additional 30 amino acid residues at the N-terminus as well as two Cys-Pro dipeptides near the C-terminus yielding a protein with 316 amino acids and a molecular mass of 36 kDa. A second potential computationally mapped isoform has been described for HO-2 as the result of alternative splicing. The sequence of this isoform differs from the canonical sequence by the lack of amino acids 1 – 26 yielding a putative protein with 287 amino acids and a predicted molecular mass of 32.8 kDa. However, no experimental confirmation is available for the shorter isoform apart from the canonical sequence.  A third isoform, termed HO-3 has been described in rats only ⁴⁰.  As the expression of HO-3 could not be detected at the mRNA level in a genomic DNA-free liver library, neither at the protein level in kidney from rats, HO-3 is supposed to be a pseudogene (HO3; Hmox2-ps1), originating from the HMOX2 mRNA ⁴⁰.

 

The model plant A. thaliana possesses three biochemically indistinguishable functional HO proteins (AtHO-1, AtHO-3, AtHO-4) and one probably inactive HO (AtHO-2) ¹⁰. All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini sufficient for chloroplast translocation. Two isoforms as the result of alternative splicing have been described for AtHO-1. The shorter isoform differs from the canonical sequence by an aa94 – 142 → D substitution. Notwithstanding the above, no experimental confirmation is available for the shorter isoform apart from the canonical sequence. Four isoforms have been reported for AtHO-2: one described alternatively spliced isoform 299 amino acids in length and three potential isoforms that are computationally mapped. Two isoforms produced by alternative splicing have been noted for AtHO-3. Isoform 2 of AtHO-3 bears a VSKKILDN → LCRYLRRY substitution at position 220 – 227 and lacks the amino acid stretch 228 – 285 of the canonical sequence. Of note, no experimental confirmation is available for the shorter isoform apart from the canonical sequence.

 

HO proteins have also been identified in prokaryotes, such as HmuO from Gram-positive Corynebacterium spec. ^{41, 42} and HemO from Gram-negative Neisseria meningitidis and N. gonorrhoeae ^{43, 44}. S. aureus was found to express two HO variants; IsdG and its paralog IsdI (64% sequence identity and 79% sequence similarity) converting heme to staphylobilin and formaldehyde ^{48, 88}. These molecules belong to the α + β protein class as part of the dimeric α + β-barrel superfamily ⁸⁹. Comparibly to S. aureus, P. aeruginosa encodes multiple HO-like enzymes such as PigA/HemO, BphO, and PhuS. While only PigA/HemO is directly involved in heme iron utilization ⁸⁴, BphO catalyzes the formation of biliverdin required for the assembly of the phytochrome-like photoreceptor BphP ⁸⁵. It is worth mentioning that BphO is expressed independently of the iron status but its expression is regulated by the cell density ⁸⁹. Several of the above mentioned microbial HOs show a high degree of homology with mammalian HO-1 ¹⁴¹. However, an independent development of certain bacterial HOs has been assumed as well ⁴⁸.

 

HOs are also expressed in pathogenic fungi as well as non-pathogenic mircoorganisms such as yeasts . The HO ortholog Hmx1p was identified in S. cerevisiae as a stress protein in response to iron deprivation ⁴⁵. In Candida albicans, Hmx1p was first reported as serving an essential function in iron assimilation from heme ⁴⁶.

 

Noteworthy, a variety of human diseases have been attributed to HMOX1/2 gene and/or protein variations. For instance, genetic variants in HMOX1 and HMOX2 have been linked to Parkinson’s disease (PD) ^142-144. HMOX1 promoter polymorphisms have been found as being associated with the susceptibility of the chronic obstructive pulmonary disease (COPD) ¹⁴⁵ and coronary heart disease ¹⁴⁶, respectively. A di-nucleotide repeat (GT)_n polymorphism located to the promoter of the HMOX1 gene has been identified as being linked to a high risk of metabolic disorders ¹⁴⁷. A complete loss of exon 2 and a two-nucleotide deletion within exon 3 was found to cause HO-1 deficiency in humans leading to severe growth retardation, persistent hemolytic anemia, severe persistent endothelial damage, as well as iron deposition in renal and hepatic tissues ^{148, 149}.