Α2β2 Tetramer Research Articles

Zinc-Substituted Hemoglobin-Albumin Cluster as a Porphyrin-Carrier for Enhanced Photodynamic Therapy.

Apohemoprotein is focused on the field of theranostics, serving as a porphyrin carrier. Hemoglobin (Hb) consists of α2β2 tetramer with iron(II)-protoporphyrin IX (heme) bound to each globin. However, heme-removed Hb (apoHb) causes dissociation at αβ interfaces and aggregation under physiological conditions. We synthesized a stable apoHb derivative comprising intramolecular-crosslinked apoHb (apoXHb) and human serum albumin (HSA), apoXHb-HSA3. ApoXHb-HSA3 engendered no aggregates in the physiological solutions. Moreover, apoXHb-HSA3 was reconstituted with zinc(II)-protoporphyrin IX (ZnP), generating ZnXHb-HSA3, a potent photosensitizer for photodynamic therapy (PDT). The photophysical properties of ZnXHb-HSA3 were identical to those of zinc-substituted XHb (ZnXHb). Cellular uptake behavior was evaluated using various cancer cell lines. ZnXHb-HSA3 released ZnP around the cells, and the free ZnP penetrated cell membranes. In contrast, protein units were not observed within the cells. ZnXHb-HSA3 showed no cytotoxicity under dark conditions and demonstrated superior PDT activity in comparison to naked ZnXHb. ZnXHb-HSA3 acts as an innovative porphyrin carrier for enhanced PDT.

Effects of unstable β-PheRS on food avoidance, growth, and development are suppressed by the appetite hormone CCHa2

ABSTRACT Amino acyl-tRNA synthetases perform diverse non-canonical functions aside from their essential role in charging tRNAs with their cognate amino acid. The phenylalanyl-tRNA synthetase (PheRS/FARS) is an α2β2 tetramer that is needed for charging the tRNAPhe for its translation activity. Fragments of the α-subunit have been shown to display an additional, translation-independent, function that activates growth and proliferation and counteracts Notch signalling. Here we show in Drosophila that overexpressing the β-subunit in the context of the complete PheRS leads to larval roaming, food avoidance, slow growth, and a developmental delay that can last several days and even prevents pupation. These behavioural and developmental phenotypes are induced by PheRS expression in CCHa2+ and Pros+ cells. Simultaneous expression of β-PheRS, α-PheRS, and the appetite-inducing CCHa2 peptide rescued these phenotypes, linking this β-PheRS activity to the appetite-controlling pathway. The fragmentation dynamic of the excessive β-PheRS points to β-PheRS fragments as possible candidate inducers of these phenotypes. Because fragmentation of human FARS has also been observed in human cells and mutations in human β-PheRS (FARSB) can lead to problems in gaining weight, Drosophila β-PheRS can also serve as a model for the human phenotype and possibly also for obesity.

A Molecular Dynamics Simulation Study of the Effects of βGln114 Mutation on the Dynamic Behavior of the Catalytic Site of the Tryptophan Synthase.

L-tryptophan (l-Trp), a vital amino acid for the survival of various organisms, is synthesized by the enzyme tryptophan synthase (TS) in organisms such as eubacteria, archaebacteria, protista, fungi, and plantae. TS, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, comprises α and β subunits that typically form an α2β2 tetramer. The enzyme's activity is regulated by the conformational switching of its α and β subunits between the open (T state) and closed (R state) conformations. Many microorganisms rely on TS for growth and replication, making the enzyme and the l-Trp biosynthetic pathway potential drug targets. For instance, Mycobacterium tuberculosis, Chlamydiae bacteria, Streptococcus pneumoniae, Francisella tularensis, Salmonella bacteria, and Cryptosporidium parasitic protozoa depend on l-Trp synthesis. Antibiotic-resistant salmonella strains have emerged, underscoring the need for novel drugs targeting the l-Trp biosynthetic pathway, especially for salmonella-related infections. A single amino acid mutation can significantly impact enzyme function, affecting stability, conformational dynamics, and active or allosteric sites. These changes influence interactions, catalytic activity, and protein-ligand/protein-protein interactions. This study focuses on the impact of mutating the βGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX1), E(A-A), and E(C3) forms of TS using the WT, βQ114A, and βQ114N versions. The results show that both the βQ114A and βQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of βGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The βQ114A mutation creates a gap between βAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting β-sites. Conversely, the βQ114N mutation positions βAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer

Collagen prolyl 4-hydroxylases (C-P4H) are α2β2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact β/PDI subunit, at 3.8Å resolution. The CAT domain interacts with the a, b', and a' domains of the β/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a β-strand, which is the edge strand of its major antiparallel β-sheet. This extra region of the CAT domain interacts tightly with the β/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a' domain and tight hydrophobic interactions with the b' domain. Using this new information, the structure of the mature C-P4H-II α2β2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2β2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman, crystallography, and DFT calculation

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or β subunit contains a ferric heme in the α2β2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.

Structural origin of cooperativity in human hemoglobin: a view from different roles of α and β subunits in the α2β2 tetramer.

This mini-review, mainly based on our resonance Raman studies on the structural origin of cooperative O2 binding in human adult hemoglobin (HbA), aims to answering why HbA is a tetramer consisting of two α and two β subunits. Here, we focus on the Fe-His bond, the sole coordination bond connecting heme to a globin. The Fe-His stretching frequencies reflect the O2 affinity and also the magnitude of strain imposed through globin by inter-subunit interactions, which is the origin of cooperativity. Cooperativity was first explained by Monod, Wyman, and Changeux, referred to as the MWC theory, but later explained by the two tertiary states (TTS) theory. Here, we related the higher-order structures of globin observed mainly by vibrational spectroscopy to the MWC theory. It became clear from the recent spectroscopic studies, X-ray crystallographic analysis, and mutagenesis experiments that the Fe-His bonds exhibit different roles between the α and β subunits. The absence of the Fe-His bond in the α subunit in some mutant and artificial Hbs inhibits T to R quaternary structural change upon O2 binding. However, its absence from the β subunit in mutant and artificial Hbs simply enhances the O2 affinity of the α subunit. Accordingly, the inter-subunit interactions between α and β subunits are nonsymmetric but substantial for HbA to perform cooperative O2 binding.

Roles of Fe-Histidine bonds in stability of hemoglobin: Recognition of protein flexibility by Q Sepharose

Using various mutants, we investigated to date the roles of the Fe-histidine (F8) bonds in cooperative O2 binding of human hemoglobin (Hb) and differences in roles between α- and β-subunits in the α2β2 tetramer. An Hb variant with a mutation in the heme cavity exhibited an unexpected feature. When the β mutant rHb (βH92G), in which the proximal histidine (His F8) of the β-subunit is replaced by glycine (Gly), was subjected to ion-exchange chromatography (Q Sepharose column) and eluted with an NaCl concentration gradient in the presence of imidazole, yielded two large peaks, whereas the corresponding α-mutant, rHb (αH87G), gave a single peak similar to Hb A. The β-mutant rHb proteins under each peak had identical isoelectric points according to isoelectric focusing electrophoresis. Proteins under each peak were further characterized by Sephadex G-75 gel filtration, far-UV CD, 1H NMR, and resonance Raman spectroscopy. We found that rHb (βH92G) exists as a mixture of αβ-dimers and α2β2 tetramers, and that hemes are released from β-subunits in a fraction of the dimers. An approximate amount of released hemes were estimated to be as large as 30% with Raman relative intensities. It is stressed that Q Sepharose columns can distinguish differences in structural flexibility of proteins having identical isoelectric points by altering the exit rates from the porous beads. Thus, the role of Fe-His (F8) bonds in stabilizing the Hb tetramer first described by Barrick et al. was confirmed in this study. In addition, it was found in this study that a specific Fe-His bond in the β-subunit minimizes globin structural flexibility.

Collagen prolyl 4-hydroxylases modify tumor progression.

Collagen is the main component of the extracellular matrix. Hydroxylation of proline residues on collagen, catalyzed by collagen prolyl 4-hydroxylase (C-P4H), is essential for the stability of the collagen triple helix. Vertebrate C-P4H is an α2β2 tetramer with three isoenzymes differing in the catalytic α-subunits, which are encoded by P4HA1, P4HA2, and P4HA3 genes. In contrast, β-subunit is encoded by a single gene P4HB. The expressions of P4HAs and P4HB are regulated by multiple cellular factors, including cytokines, transcription factors, and microRNAs. P4HAs and P4HB are highly expressed in many tumors and participate in cancer progression. Several inhibitors of P4HAs and P4HB have been confirmed to have anti-tumor effects, suggesting that targeting C-P4H is a feasible strategy for cancer treatment. Here, we summarize recent progresses on the function and expression of regulatory mechanisms of C-P4H in cancer progression and point out the potential development of therapeutic strategies in targeting C-P4H in the future.

β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies.

Hemoglobin is a tetrameric protein composed of two α and two β chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and β-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two β subunits (α2β2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in β-globin production cause two of the most common monogenic-inherited disorders: β-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of β-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.

Structural enzymology binding studies of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase (type-II): High affinity peptides have a PxGP sequence motif.

The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α2 β2 tetramer) binds proline-rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C-P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C-terminal domain of the α subunit). The crystal structures of the PSB domain of the human C-P4H isoform II (PSB-II) complexed with and without various short proline-rich peptides are described. The comparison with the previously determined PSB-I peptide complex structures shows that the C-P4H-I substrate peptide (PPG)3 , has at most very weak affinity for PSB-II, although it binds with high affinity to PSB-I. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB-II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB-I peptide complexes. In these PSB-II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide-binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB-I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline-rich peptide sequences that bind with high affinity to PSB-II.

Discrimination Between Infant and Adult Bloodstains Using Micro-Raman Spectroscopy: A Preliminary Study.

In the present study, we used micro-Raman spectroscopy with high-resolution analysis to discriminate between bloodstains from infants and bloodstains from adults. Raman peaks were detected at 674, 754, 976, 1002, 1105, 1127, 1176, 1248, 1340, 1368, 1390, 1560, and 1611cm-1 ; these peaks were derived from hemoglobin, albumin, and glucose. However, a peak was obtained at 1105cm-1 , which was assigned to histidine; this peak was observed only for bloodstains from adults. Human adult hemoglobin (HbA) is composed of an α2 β2 tetramer structure, whereas human fetal hemoglobin (HbF) is composed of an α2 γ2 . Therefore, the lack of a Raman peak at 1105cm-1 in bloodstains from infants indicates the possibility of two histidine substitutions (His116Ile and His143Ser) in the γ chain of HbF. This study discriminates between bloodstains from infants and bloodstains from adults using micro-Raman spectroscopy, with beneficial implications in forensic science.

Analysis of Dimeric αβ Subunit Exchange between PEGylated and Native Hemoglobins (α2β2 Tetramer) in an Equilibrated State by Intramolecular ββ-Cross-Linking.

Various chemical modifications of hemoglobin (Hb) including PEGylation have been investigated to produce red blood cell substitutes. Some of those modifications are designed on the premise that the α2β2 tetrameric structure of Hb is fundamentally stable and that it rarely dissociates into two αβ dimers in a physiological condition. However, in the present work using the "clipping" method we detected and quantitatively analyzed the considerable degree of exchange reaction of αβ subunits between β93Cys-bis-PEGylated and native Hbs through dissociation into αβ dimers and restructuring to α2β2 tetramer in a physiological condition. The equilibrium constant ( Keq) of subunit exchange reactions increased from 0.82 to 2.86 with increasing molecular weight of PEG from 2 to 40 kDa, indicating that longer PEG chains enhanced such exchange reaction. The results suggest that the exchange might occur for other modified Hbs even at a practically high concentration for use as a red blood cell substitute.

Heterogeneity between Two α Subunits of α2β2 Human Hemoglobin and O2 Binding Properties: Raman, 1H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the β subunit of α2β2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)β(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the β subunit: Hb M Hyde Park (β92His → Tyr), Hb M Saskatoon (β63His → Tyr), and Hb M Milwaukee (β67Val → Glu). In contrast with the β subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2β2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)β(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)β(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)β(Fe3+)β(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2β2 tetramer changes the other α subunit to the R structure.

α-Synuclein is a Novel Microtubule Dynamase.

α-Synuclein is a presynaptic protein associated to Parkinson’s disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson’s disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

The Most Interesting Enzyme in the World

Ribonucleotide reductases of the class I family are α2β2 tetramers. Like all RNRs they are subject to allosteric control mechanisms affecting activity and specificity. In this issue of Structure, Johansson etal. (2016) present a structural analysis of an unusual mode of activity site regulation.

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer.

Human hemoglobin (Hb), which is an α2β2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by ‘cooperativity’. This property is indispensable for its function of O2 transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O2-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O2-binding, but its absence in the β subunit simply enhances the O2-affinity of α subunit.

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.

Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen

Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens.Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets.In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification.

Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.