Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin (original) (raw)
Related papers
Structure of hemocyanin from garden snail Helix lucorum
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2010
Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as β-HlH, α D -HlH and α N -HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (β-HlH) and 1079 kDa (α D -HlH, and α N -HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl 2 . All isoforms were reassociated into didecamers and tubules with different length, but in contrast to α D -HlH isoform, longer tubules were observed in β-HlH. Moreover the structure of β-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.
cDNA sequence of three isoforms of the hemocyanin from Helix vulgaris
Gene
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.
Isolation and spectroscopic characterization of the structural subunits of keyhole limpet hemocyanin
Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67³C and the free energy of stabilization in water, v G H 2 O D , towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence. ß
Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 2000
The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy-and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, T c , determined by fluorescence spectroscopy, are in the region of 50-60°C, coinciding with the melting temperatures, T m , determined by CD spectroscopy. The free energy of stabilization in water, DG D H2O , toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.
The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum
Gene, 2011
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.
European Journal of Biochemistry, 1997
Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH ; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLHl and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLHl subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45 -60 kDa functional domains (termed l a through lh) and determined their sequential arrangement. The KLHl domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain lh, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLHl and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional l h domain. It was also shown that the KLHl didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Triskaline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl, and 5 mM MgCI,, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLHl multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.
Structural and conformational stability of hemocyanin from the garden snail Cornu aspersum
Zeitschrift für Naturforschung C, 2019
Various aspects of biomedical applications of molluscan hemocyanins, associated with their immunogenic properties and antitumor activity, promoted us to perform structural studies on these glycoproteins. The stability and reassociation behavior of native Cornu aspersum hemocyanin (CaH) are studied in the presence of different concentrations of Ca 2+ and Mg 2+ ions and pH values using electron microscopy. Higher concentrations of those ions led to a more rapid reassociation of CaH, resulting in stable multidecamers with different lengths. The conformational changes of native CaH are investigated within a wide pH-temperature range by UV circular dichroism. The relatively small changes of initial [θ] λ indicated that many secondary structural elements are preserved, even at high temperatures above 80 °C, especially at neutral pH. The mechanism of thermal unfolding of CaH has a complicated character, and the process is irreversible. The conformational stability of the native didecameric aggregates of CaH toward various denaturants indicates that hydrophilic and polar forces stabilize the quaternary structure. For the first time, the unfolding of native CaH in water solutions in the presence of four different denaturants is investigated. The free energy of stabilization in water, ∆G D H 2 O , was calculated in the range of 15.48-16.95 kJ mol −1. The presented results will facilitate the further investigation of the properties and potential applications of CaH.
Journal of Biological Chemistry, 2008
We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca 2؉ and/or Mg 2؉ in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4°C, increases at 37°C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg 2؉ is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.
Proceedings of the Bulgarian Academy of Sciences
Glycosylation plays an important role in many important processes in organisms. Their participation in the construction of the tertiary structure and immunotherapeutic activity of the hemocyanin Rapana venosa (RvH) from the Black Sea has been presented. Based on these results, the importance of glycans in organization of the structure of hemocyanin from the abdominal mollusk Haliotis tuberculata (HtH) was also studied. After incubation of both isoforms HtH1 and HtH2 with yeast Saccharomyces cerevisiae and enzyme Zymolyase, fractions with lower molecular weight (MW) between 45 and 65 kDa, which corresponds to MW of functional units (FUs) of hemocyanins, was identified. A comparative analysis of 10% SDS PAGE assays confirmed the depolymerization of the subunits HtH1 and HtH2 due to the specific glycosidase in the yeast S. cerevisiae and the enzyme Zymolyase. The presented results confirm our hypothesis that glycans are involved in the formation of the structural subunits of HtH.