Biochemical and molecular characterisation of hemocyanin from the amphipod <Emphasis Type="Italic">Gammarus roeseli</Emphasis>: complex pattern of hemocyanin subunit evolution in Crustacea

Hemocyanin is a copper-containing respiratory protein that is widespread within the arthropod phylum. Among the Crustacea, hemocyanins are apparently restricted to the Malacostraca. While well-studied in Decapoda, no hemocyanin sequence has been known from the ’lower’ Malacostraca. The hemocyanin of the amphipod Gammarus roeseli is a hexamer that consists of at least five distinct subunits. The complete cDNA sequence of one subunit and a tentative partial sequence of another subunit have been determined. The complete G. roeseli hemocyanin subunit comprises 2,150 bp, which translates in a protein of 672 amino acids with a molecular mass of 76.3 kDa. Phylogenetic analyses show that, in contrast to previous assumptions, the amphipod hemocyanins do not belong to the α-type of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to α and γ-subunits most likely at the time of separation of peracarid and eucarid Crustacea about 300 million years ago. Molecular clock analyses further suggest that the divergence of β-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA) in the malacostracan stemline, while α- and γ-type subunits separated 258 MYA, and pseudohemocyanins and γ-subunits 210 million years ago.     

The oxygen-binding modulation of hemocyanin from the Southern spiny lobster <Emphasis Type="Italic">Palinurus gilchristi</Emphasis>

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 × 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 × 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin–oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0–1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium–hemocyanin interaction, three other species belonging to Palinura.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

The First Complete cDNA Sequence of the Hemocyanin from a Bivalve,the Protobranch <Emphasis Type="Italic">Nucula nucleus</Emphasis>

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M _r = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M _r = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million years ago, in conformity with fossil records from the late Cambrian. [Reviewing Editor: Dr. Rüdiger Cerff] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1 and AJ786640 for NnH2.     

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with 10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.     

Diversity of stonefly hexamerins and implication for the evolution of insect storage proteins

Hexamerins are large storage proteins of insects in the 500 kDa range that evolved from the copper-containing hemocyanins. Hexamerins have been found at high concentration in the hemolymph of many insect taxa, but have remained unstudied in relatively basal taxa. To obtain more detailed insight about early hexamerin evolution, we have studied hexamerins in stoneflies (Plecoptera). Stoneflies are also the only insects for which a functional hemocyanin is known to co-occur with hexamerins in the hemolymph. Here, we identified hexamerins in five plecopteran species and obtained partial cDNA sequences from Perla marginata (Perlidae), Nemoura sp. (Nemouridae), Taeniopteryx burksi (Taeniopterygidae), Allocapnia vivipara (Capniidae), and Diamphipnopsis samali (Diamphipnoidae). At least four distinct hexamerins are present in P. marginata. The full-length cDNA of one hexamerin subunit was obtained (PmaHex1) that measures 2475 bp and translates into a native polypeptide of 702 amino acids. Phylogenetic analyses showed that the plecopteran hexamerins are monophyletic and positioned at the base of the insect hexamerin tree, probably diverging about 360 million years ago. Within the Plecoptera, distinct hexamerin types evolved before the divergence of the families. Mapping amino acid compositions onto the phylogenetic tree shows that the accumulation of aromatic amino acids (and thus the evolution of "arylphorins") commenced soon after the hexamerins diverged from hemocyanins, but also indicates that hexamerins with distinct amino acid compositions reflect secondary losses of aromatic amino acids.     

Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.     

Subunit sequences of the 4 x 6-mer hemocyanin from the golden orb-web spider, Nephila inaurata.

The transport of oxygen in the hemolymph of many arthropod and mollusc species is mediated by large copper-proteins that are referred to as hemocyanins. Arthropod hemocyanins are composed of hexamers and oligomers of hexamers. Arachnid hemocyanins usually form 4 x 6-mers consisting of seven distinct subunit types (termed a-g), although in some spider taxa deviations from this standard scheme have been observed. Applying immunological and electrophoretic methods, six distinct hemocyanin subunits were identified in the red-legged golden orb-web spider Nephila inaurata madagascariensis (Araneae: Tetragnathidae). The complete cDNA sequences of six subunits were obtained that corresponded to a-, b-, d-, e-, f- and g-type subunits. No evidence for a c-type subunit was found in this species. The inclusion of the N. inaurata hemocyanins in a multiple alignment of the arthropod hemocyanins and the application of the Bayesian method of phylogenetic inference allow, for the first time, a solid reconstruction of the intramolecular evolution of the chelicerate hemocyanin subunits. The branch leading to subunit a diverged first, followed by the common branch of the dimer-forming b and c subunits, while subunits d and f, as well as subunits e and g form common branches. Assuming a clock-like evolution of the chelicerate hemocyanins, a timescale for the evolution of the Chelicerata was obtained that agrees with the fossil record.     

Molecular evolution of the arthropod hemocyanin superfamily

Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from these enzymes before the radiation of the extant euarthropodan subphyla. Due to different functional constraints, replacement rates greatly vary between the clades. Divergence times were thus estimated assuming local molecular clocks using several substitution models. The results were consistent and indicated the separation of the cheliceratan and crustacean hemocyanins close to 600 MYA. The different subunit types of the multihexameric cheliceratan hemocyanin have a rather conservative structure and diversified in the arachnidan stemline between 550 and 450 MYA. By contrast, the separation of the crustacean (malacostracan) hemocyanin subunits probably occurred only about 200 MYA. The nonrespiratory pseudohemocyanins evolved within the Decapoda about 215 MYA. The insect hemocyanins and storage hexamerins emerged independently from the crustacean hemocyanins. The time of divergence of the insect proteins from the malacostracan hemocyanins was estimated to be about 430-440 MYA, providing support for the notion that the Hexapoda evolved from the same crustacean lineage as the Malacostraca.     

Putative phenoloxidases in the tunicate<Emphasis Type="Italic"> Ciona intestinalis</Emphasis> and the origin of the arthropod hemocyanin superfamily

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157–2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.Abbreviations EST expressed sequence tags Communicated by G. Heldmaier     

Molecular cloning and characterization of <Emphasis Type="Italic">GhAPm</Emphasis>, a gene encoding the μ subunit of the clathrin-associated adaptor protein complex that is associated with cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) fiber development

The clathrin-associated adaptor protein (AP) complexes are the primary clathrin adaptors that contribute to the formation of clathrin-coated vesicles (CCVs). The GhAPm gene (GenBank accession number: GU359054), which encodes the medium subunit of the AP complexes, was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 1590 bp in size and encoded an open reading frame (ORF) of 416 amino acids with a molecular weight of 46 kDa. The GhAPm protein shared 81–85% identity at the amino acid level with the AP complex μ subunits isolated from Vitis vinifera, Glycine max, Populus trichocarpa, Ricinus communis and Arabidopsis thaliana, respectively. The corresponding genomic DNA, containing eight exons and seven introns, was isolated and analyzed. Also, a 5′-flanking region was analyzed, and a group of putative cis-acting elements were identified. DNA gel blot analysis showed that there is only one GhAPm gene in the cotton genome. Real-time RT-PCR analysis revealed that GhAPm is expressed in the root, stem, leaf, petal, ovule, and fiber. However, the interesting finding is that GhAPm expression level was shown to increase steadily as the cotton fiber develops. In 30 DPA fibers, expression increases sharply and arrives at a peak then the expression levels decrease rapidly. Based on these data, we propose that GhAPm has a critical role in cotton membrane trafficking and fiber development.     

Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components.

The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.     

Molecular characterisation and evolution of the hemocyanin from the European spiny lobster,<Emphasis Type="Italic"> Palinurus elephas</Emphasis>

The hemocyanin of the European spiny lobster Palinurus elephas (synonym: Palinurus vulgaris) is a hexamer composed by four closely related but distinct subunits. We have obtained the full cDNA sequences of all four subunits, which cover 2275-2298 bp and encode for native polypeptides of 656 and 657 amino acids. The P. elephas hemocyanin subunits belong to the alpha-type of crustacean hemocyanins, whereas beta- and gamma-subunits are absent in this species. An unusual high ratio of non-synonymous versus synonymous nucleotide substitutions was observed, suggesting positive selection among subunits. Assuming a constant evolution rate, the P. elephas hemocyanin subunits emerged from a single hemocyanin gene around 25 million years ago. The alpha-type hemocyanins of P. elephas and the American spiny lobster Panulirus interruptus split around 100 million years ago. This is about five times older than the assumed divergence time of the species and suggests that the genera may have split with the formation of the Atlantic Ocean. The application of the Bayesian method for phylogenetic inference allows for the first time a solid reconstruction of the evolution of the decapod hemocyanins, showing that the beta-subunit types diverged first and that the crustacean pseudo-hemocyanins are associated with the gamma-type subunits.     

The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M_r = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M_r = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian. [Reviewing Editor: Dr. Axel Meyer] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ619741.     

Cloning,characterization, expression,and copper sensitivity of the metallothionein-1 gene in the Chinese mitten crab, <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

A full-length metallothionein-1(MT-1) cDNA was cloned from the Chinese mitten crab, Eriocheir sinensis, based upon the hepatopancreas cDNA library. The full-length cDNA contained a single 180 bp open reading frame that encoded a 59 amino acid protein. The deduced amino acid sequence was cysteine (Cys)-rich, with residues observed in patterns characteristic of other reported MTs: Cys–X–Cys, Cys–X–X–Cys, or Cys–X–X–X–Cys. Gene structure obtained via PCR yielded a 3816 bp gene, which was comprised of three exons and two introns arranged in a “3 + 2” pattern. The cloned 5′flanking region (1,735 bp) contained several predicted binding sites, which included MREs, AP-1, SP1, USF, GATA, HNF-1, and HSF. MT-1 mRNA expression analysis revealed that while levels were highest in the hepatopancreas, expression was abundant in testis and thoracic ganglia, moderate in intestine (P < 0.05), and weak in other tissues (P < 0.05). MT-1 mRNA expression exhibited reproductive variation in the male, with levels approximately tenfold greater in August, during seasonal gonadal maturation, compared to other times of the year. Cu²⁺ exposure via tank water (0–1 mg/l for 7 days) resulted in a dose-dependent bell curve response in MT-1 mRNA expression, with peak expression observed after exposure to 0.1 mg/l Cu²⁺. A time course experiment (0.1 mg/l Cu²⁺ over 9 days) revealed MT-1 mRNA expression peaked sharply on day 5 before gradually decreasing with prolonged exposure. In the present report, we provide sequence analysis of the first MT-1 gene cloned in E. sinensis, and evidence that its physiological and toxicological regulation is evolutionary conserved.     

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of <Emphasis Type="Italic">Dolichos lablab</Emphasis>

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively. The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional unit of the Rapana thomasiana (grosse) (marine snail, gastropod) hemocyanin

The amino terminal functional unit (domain a) of the Rapana hemocyanin “heavy” structural subunit, designated as Rta, was obtained after limited trypsinolysis of the whole polypeptide chain. Mass spectrometric analysis showed a molecular mass of 49,698 daltons for the electrophoretically homogeneous fragment. Twenty-five amino acid residues were sequenced directly from the N-terminus of Rta, which allowed the location of the domain in the polypeptide chain of the subunit. Physicochemical parameters were determined by absorption and fluorescence spectroscopy and circular dichroism. Comparison with the respective parameters of the whole Rapana hemocyanin showed that the polypeptide backbone folding, binuclear active site and capability of oxygen binding of the isolated functional unit are identical to those of the native hemocyanin. Comparison of N-terminal sequences of functional units from different molluskan hemocyanins and located at different positions revealed some evolutionary relationships.