Haptoglobin, a hemoglobin-binding plasma protein, is present in bony fish and mammals but not in frog and chicken

Abstract

Hemoglobin (Hb) released from erythrocytes may cause oxidation of lipids and proteins. Haptoglobin (Hp), which occurs in the plasma of all mammals, binds free Hb and inhibits its oxidative activity. It is not known whether this protective protein also exists in lower vertebrates. By analyzing available genomic sequences, we have found that bony fish, but not more primitive animals, have a gene coding for a protein homologous to mammalian Hp. Furthermore, we show that this protein is present in the plasma of Japanese pufferfish (Takifugu rubripes) and that it binds Hb. These results, together with a phylogenetic analysis, suggest that Hp evolved from a complement-associated protein (mannose-binding lectin-associated serine proteinase, MASP), with the emergence of fish. Surprisingly, we found that both chicken (Gallus gallus) and the Western clawed frog (Xenopus tropicalis) lack the Hp gene. In chicken plasma, however, we identified a different type of Hb-binding protein, PIT54, which has been reported to be a potent antioxidant. PIT54 is a soluble member of the family of scavenger receptor cysteine-rich proteins, and we found that its gene exists only in birds. We also show that the plasma of ostrich (Strutio camelus), a primitive bird, contains both PIT54 and Hp. Collectively, our data suggest that PIT54 has successively taken over the function of Hp during the evolution of the avian lineage and has completely replaced the latter protein in chicken.

Fig. 1.

Amino acid sequence alignment of human Hp (gi: 1620396) and HpLs of three bony fishes: T. nigroviridis, T. rubripes, and D. rerio. Amino acid residues corresponding to the β-chain of human Hps are shaded. Identical residues, conserved substitutions, and semiconserved substitutions are indicated by ∗, :, and ·, respectively. The signal peptide of human Hp and the putative signal sequences of the fish proteins are in bold. (The HpL genes of T. nigroviridis and T. rubripes code for an additional 90–100 aa upstream of the predicted signal peptides. Analysis of the Kozak sequences around the possible initiation codons shows that the second methionine codon is more likely to be used for initiating translation.) Consensus cleavage sequences for subtilisin-like proprotein convertases in the HpLs and the corresponding region in human Hp are framed. Conserved cysteine residues are indicated by arrows. Amino acids substituting the essential serine and histidine residues in the active site of homologous SPs are underlined. The bar over the alignment marks the amino acid sequence corresponding to the histidine loop of SPs.

Fig. 2.

Identification of Hb-binding proteins in serum of T. rubripes. Serum (Con) was incubated with Hb-containing gel beads. After centrifugation, unbound proteins were removed (Sup) and bound proteins were eluted with 8 M urea (Eluate). All samples were then analyzed by SDS/PAGE under reducing conditions followed by staining. Protein bands marked 1–4 were subjected to MS. The molecular masses in kDa of reference proteins are shown to the left.

Fig. 3.

Identification of Hb-binding proteins in ostrich serum. (A) Ostrich serum (Con) was incubated with Hb-containing gel beads. After centrifugation, unbound proteins were removed (Sup), and bound proteins were eluted with increasing concentrations of urea (Eluate). All samples were then analyzed by SDS/PAGE under reducing or nonreducing conditions, followed by staining. Protein bands marked 1–6 are described in the text. The molecular masses in kDa of reference proteins are shown to the left. (B) Protein from band 1 was analyzed by liquid chromatography tandem MS yielding the sequences of several peptides. The alignment of these peptides with the known sequence of chicken PIT54 is shown. Peptides aligning at multiple places are underlined. Amino acid residues in locations common to PIT54 and the peptides are in bold.

Fig. 4.

Phylogenetic analysis using the maximum-parsimony method of human Hp, fish HpLs, and related human and fish proteinases. The amino acid sequences of the SP domain of the following proteins were aligned by using CLUSTALW and then manually corrected: human (HSa), Hp (gi: 1620396), C1r (gi: 115204), C1s (gi: 115205), MASP-1 (gi: 19860140), MASP-2 (gi: 7387859), and MASP-3 (gi: 15088517); MASP-1 (gi: 30089299), MASP-2a (gi: 26106026), and MASP-2b (gi: 30089301) from Lethenteron japonicum (LJa); HpL, MASP-2 (gi: 49354669), C1r/sA (gi: 11177022), and C1r/sB (gi: 11177024) from Cyprinus carpio (CCa); MASP (gi: 6407545) from Triakis scyllium (TSc); HpL, C1r/s (gi: 40217256) from Oncorhynchus mykiss (OMy); HpL, C1r/sA* (ensembl: SINFRUP00000156151), C1r/sB* (ensembl: SINFRUP00000174510), and MASP-2* (ensembl: SINFRUP00000139601) from T. rubripes (TFu); HpL, MASP-2* (ensembl: GSTENT00021129001), C1r/sA* (ensembl: GSTENT00015325001), and C1r/sB* (ensembl: GSTENT00015326001) from T. nigroviridis (TNi); HpL, MASP2a* (ensembl: ENSDARP00000049759), MASP2b* (ensembl: ENSDARP00000028217), and C1r/s* (ensembl: ENSDARP00000017015) from D. rerio (DRe). The names of protein sequences indicated by a star were assigned based on the following rules: C1r/s for the proteinases that were most similar to either mammalian C1r or C1s and MASP-2 for the proteinases most similar to mammalian MASPs and lacking the histidine-loop cysteins (see text). An unrooted phylogenetic tree was constructed by using the maximum-parsimony method. Numbers at nodes denote bootstrap values with 1,000 replicates. The Hp, MASP, and C1r/s families of proteins are circled.

Fig. 5.

Synteny map of human Hp, fish HpL of T. nigroviridis, T. rubripes, and D. rerio loci, and the corresponding regions of amphibian X. tropicalis and chicken (G. gallus) genome, according to the ensembl program (www.ensembl.org). Hpr, Hp-related; DHX38, ATP-dependent RNA helicase; TXNL4B, thioredoxin-like protein 4B; ST5A1, sulfotransferase 5A1; DPEP1, dehydropeptidase 1; PKD1L3, polycystic kidney disease 1-like 3; ?, genes of unknown function.

Fig. 6.

Possible course of HpL and Hp evolution. MASP proteins and C1r and C1s consist of two C1r/C1s/Uegf/bone morphogenic protein 1 (CUB) domains, one epidermal growth factor (EGF) domain, two CCP domains, and one SP domain (details in text). Through partial duplication (I) of a gene coding for a Hp, C1r, C1s, and MASP-2 ancestor [anc(MASP-2/C1r/C1s/Hp)], a gene coding for a Hp prototype (prtHp) arose. Subsequently, mutations of the essential His (H) and Ser (S) residues in the active site of the SP domain (II) gave rise to a proteolytically inactive protein (ancHp). In fish, the CCP domain was then deleted (III), and an intron was inserted into the exon coding for the SP domain (IV), resulting in the appearance of present-day HpL. Mammalian Hp retained the CCP and the SP domain. Solid and dashed lines under the SP domain represent exons and introns, respectively, of the gene fragment coding for this domain.