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J Markl

Publications and source records attributed to J Markl.

At least 19 recordsLinked to original sources
PubMed2000

Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy.

A 12 A resolution three-dimensional density map of the Haliotis tuberculata hemocyanin type 1 (HtH1) didecamer has been obtained by cryoelectron microscopy of unstained molecules and angular reconstitution. The dyad symmetry of the 8 MDa D5 HtH1 didecamer, formed by the pairing of two asymmetric 4 MDa ring-like C5 decamers, is emphasised. The major and minor surface helical grooves of the didecamer are well defined, in agreement with earlier data on molluscan hemocyanins. The location of the obliquely orientated repeating unit, a subunit dimer, within the decamer has been defined. Following interactive extraction of this dimer, several new structural features of the dimer and of the subunit have now emerged with improved detail. The subunit dimer possesses pseudo 2-fold symmetry, resulting from the steric arrangement of the wall domains/functional units (FUs-abcdef) of the two subunits. The arc and collar FUs (g and h) depart from this inherent 2-fold symmetry and are thereby responsible for the asymmetry of the C5 decamer, with the internalised collar/arc complex at one edge of the decamer. The FU heterodimers forming the wall morphological units have a hollow centre, and thus create a series of repeating channels that extend within the wall through all three tiers of the decamer. The connections between the wall and the arc are defined with improved clarity, and evidence is provided to indicate that the arc and collar FU pairs have a homodimeric composition (gg and hh, respectively). Two possibilities for the subunit path within the subunit dimer are presented, which correlate with the available structural, immunolabelling and protease cleavage data from HtH1 and other molluscan hemocyanins.

Animals↗
PubMed2000

The sequence of a gastropod hemocyanin (HtH1 from Haliotis tuberculata).

The eight functional units (FUs), a-h, of the hemocyanin isoform HtH1 from Haliotis tuberculata (Prosobranchia, Archaeogastropoda) have been sequenced via cDNA, which provides the first complete primary structure of a gastropod hemocyanin subunit. With 3404 amino acids (392 kDa) it is the largest polypeptide sequence ever obtained for a respiratory protein. The cDNA comprises 10,758 base pairs and includes the coding regions for a short signal peptide, the eight different functional units, a 3'-untranslated region of 478 base pairs, and a poly(A) tail. The predicted protein contains 13 potential sites for N-linked carbohydrates (one for HtH1-a, none for HtH1-c, and two each for the other six functional units). Multiple sequence alignments show that the fragment HtH1-abcdefg is structurally equivalent to the seven-FU subunit from Octopus hemocyanin, which is fundamental to our understanding of the quaternary structures of both hemocyanins. Using the fossil record of the gastropod-cephalopod split to calibrate a molecular clock, the origin of the molluscan hemocyanin from a single-FU protein was calculated as 753 +/- 68 million years ago. This fits recent paleontological evidence for the existence of rather large mollusc-like species in the late Precambrian.

Amino Acid Sequence↗
PubMed2000

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2.

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.

Animals↗
PubMed2000

Keyhole limpet hemocyanin: molecular structure of a potent marine immunoactivator. A review.

OBJECTIVES: In this short review we present a survey of the available biochemical and electron microscopic data on keyhole limpet hemocyanin (KLH). RESULTS: The biosynthesis of KLH and its biological role are discussed and the purification of the two isoforms of KLH (KLH1 and KLH2) presented in some detail. The determination of the molecular mass of KLH, its functional unit structure, carbohydrate content, immunological analysis and aspects of the molecular biology of KLH are all dealt with. Transmission electron microscopy (TEM) and crossed immunoelectrophoresis have played a significant part in the understanding of KLH structure. We present a summary of TEM studies on the native oligomers of KLH, the experimental manipulation of the different oligomeric states, immunological analysis and subunit reassociation. CONCLUSION: This fundamental structural information provides the scientific background upon which the understanding of the in vivo immunostimulatory function of KLH can be based.

Adjuvants, Immunologic↗
PubMed1999

Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h.

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.

Amino Acid Sequence↗
PubMed1999

Hemocyanin subunit organization of the gastropod Rapana thomasiana.

RtH1 and RtH2, the two hemocyanin isoforms of the prosobranch gastropod Rapana thomasiana, have been purified by anion-exchange chromatography and studied by SDS-PAGE and immunoelectrophoresis. Both subunit types are built up of eight functional units (FUs). Under reducing conditions subunit RtH2 splits into two fragments, RtH2-a-f and RtH2-gh, suggesting the presence of a disulfide bridge between FU2-f and FU2-g. By proteolytic cleavage of the subunits into three-, two-, and single-FU fragments, purification of fragments by HPLC, N-terminal sequencing of the peptides, and crossed-line immunoelectrophoresis, FUs-a-h of RtH2 and FU-a, FU-d, FU-e, and FU-f of RtH1 were identified and correlated to the eight-FUs pattern of immunoelectrophoresis. FU-a, FU-e, and FU-f of RtH1 and RtH2 are very closely related immunologically. RtH1 and RtH2 both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the prosobranch gastropod Megathura crenulata.

Amino Acid Sequence↗
PubMed1999

Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH).

The complete amino acid sequence of the Megathura crenulata hemocyanin functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% beta-sheet and 29% alpha-helix contents. A multiple sequence alignment with other molluscan hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural equivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calculated from its sequence and a measured mass of approx. 56 kDa; the mass difference is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no potential N-linked carbohydrate attachment sites, and this was supported by its inability to bind concanavalin A. Also KLH1-c was unreactive, whereas most, if not all, other functional units of KLH1 and KLH2 reacted positively to this lectin. On the other hand, peanut agglutinin specifically binds KLH2-c, indicating the presence of O-glycosidically linked carbohydrates in this functional unit. This contrasts to all other KLH functional units (including KLH1-c), which lack O-linked glycosides. The present results are discussed in view of the recent X-ray structure of the functional unit g from Octopus hemocyanin, and a published record of the Thomsen Friedenreich tumor antigenic epitope in KLH.

Amino Acid Sequence↗
PubMed1999

Identification, structure, and properties of hemocyanins from Diplopod myriapoda.

Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely arranged in a 36-mer (6 x 6) native molecule. It has a high oxygen affinity (P(50) = 4.7 torr) but low cooperativity (h = 1.3 +/- 0.2). Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon, Scutigera coleoptrata (Chilopoda), indicating a rather conservative architecture of the myriapod hemocyanins. Western blotting demonstrates shared epitopes of Spirostreptus hemocyanin with both chelicerate and crustacean hemocyanins, confirming its identity as an arthropod hemocyanin.

Animals↗
PubMed1999

Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octopus dofleini hemocyanin. HtH2-e is the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequences are compared to their counterparts in Octopus, Helix pomatia and HtH1 (from the latter, the sequences of FU-f, FU-g and FU-h have recently been determined) and discussed in relation to the recent 2.3 A X-ray structure of FU-g from Octopus hemocyanin and the 15 A three-dimensional reconstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamers in vivo, the additional N-glycosylation sites in HtH1 compared to HtH2 are discussed.

Amino Acid Sequence↗
PubMed1999

Identification of four distinct subunit types in the unique 6 x 6 hemocyanin of the centipede Scutigera coleoptrata.

We isolated 6 x 6 hemocyanin, dissociated it into subunits, and examined it by electron microscopy. The subunits were separated by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate PAGE, and crossed immunoelectrophoresis. Single subunits were isolated by gel cutting from native PAGE and identified as hemocyanin by measuring their ultraviolet spectrum. A total of four distinct hemocyanin subunits were identified, and the subunit pattern of the three electrophoresis systems assigned to each other. The relative proportion of subunits a:b:c:d were 2 : 2 :>: 1 as determined by densitometry. Presumably, c and d act as linkers between hexamers.

Animals↗
PubMed1999

Keyhole limpet hemocyanin (KLH): a biomedical review.

In this review we present a broad survey of fundamental scientific and medically applied studies on keyhole limpet hemocyanin (KLH). Commencing with the biochemistry of KLH, information on the biosynthesis and biological role of this copper-containing respiratory protein in the marine gastropod Megathura crenulata is provided. The established methods for the purification of the two isoforms of KLH (KLH1 and KLH2) are then covered, followed by detailed accounts of the molecular mass determination, functional unit (FU) structure, carbohydrate content, immunological analysis and recent aspects of the molecular genetics of KLH. The transmission electron microscope (TEM) has contributed significantly to the understanding of KLH structure, primarily from negatively stained images. We give a brief account of TEM studies on the native KLH oligomers, the experimental manipulation of the oligomeric states, together with immunolabelling data and studies on subunit reassociation. The field of cellular immunology has provided much relevant biomedical information on KLH and has led to the expansion of use of KLH in experimental immunology and clinically as an immunotherapeutic agent; this area is presented in some detail. The major clinical use of KLH is specifically for the treatment of bladder carcinoma, with efficacy probably due to a cross-reacting carbohydrate epitope. KLH also has considerable possibilities for the treatment of other carcinomas, in particular the epithelially derived adenocarciomas, when used as a carrier for carcinoma ganglioside and mucin-like epitopes. The widespread use of KLH as a hapten carrier and generalised vaccine component represent other major on-going aspects of KLH research, together with its use for the diagnosis of Schistosomiasis, drug assay and the treatment of drug addiction. Immune competence testing, assessment of stress and the understanding of inflammatory conditions are other areas where KLH is also making a useful contribution to medical research.

Adjuvants, Immunologic↗
PubMed1999

Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1). Organization of the approximately 400 kDa subunit, and amino acid sequence of its functional units f, g and h.

We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a approximately 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemocyanin types of the closely related marine gastropod Megathura crenulata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, SDS/PAGE and N-terminal sequencing, we identified eight different 40-60 kDa functional units in HtH1, termed HtH1-a to HtH1-h, and determined their linear arrangement within the elongated subunit. From Haliotis mantle tissue, rich in hemocyanin-producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH-specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C-terminal functional units f, g and h of HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit f and functional unit g from the cephalopod Octopus dofleini. HtH1-f, which is the first sequenced functional unit of type f from a gastropod hemocyanin, corresponds to functional unit f from Octopus. Also functional unit g from Haliotis and Octopus correspond to each other. HtH1-h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of approximately 95 amino acids, which is lacking in functional units a to g and aligns with a published peptide sequence of 48 amino acids from functional unit h of Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus, the 15 A three-dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 A X-ray structure of functional unit g from Octopus hemocyanin.

Amino Acid Sequence↗
PubMed1998

Biochemical identification and tissue-specific expression patterns of keratins in the zebrafish Danio rerio.

We have identified a number of type I and type II keratins in the zebrafish Danio rerio by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot-binding assay and immunoblotting. These keratins range from 56 kDa to 46 kDa in molecular mass and from pH 6.6 to pH 5.2 in isoelectric point. Type II zebrafish keratins exhibit significantly higher molecular masses (56-52 kDa) compared with the type I keratins (50-48 kDa), but the isoelectric points show no significant difference between the two keratin subclasses (type II: pH 6.0-5.5; type I: pH 6.1-5.2). According to their occurrence in various zebrafish tissues, the identified keratins can be classified into "E" (epidermal) and "S" (simple epithelial) proteins. A panel of monoclonal anti-keratin antibodies has been used for immunoblotting of zebrafish cytoskeletal preparations and immunofluorescence microscopy of frozen tissue sections. These antibodies have revealed differential cytoplasmic expression of keratins; this not only includes epithelia, but also a variety of mesenchymally derived cells and tissues. Thus, previously detected fundamental differences in keratin expression patterns between higher vertebrates and a salmonid, the rainbow trout Oncorhynchus mykiss, also apply between vertebrates and the zebrafish, a cyprinid. However, in spite of notable similarities, trout and zebrafish keratins differ from each other in many details. The present data provide a firm basis from which the application of keratins as cell differentiation markers in the well-established genetic model organism, the zebrafish, can be developed.

Animals↗
PubMed1998

Tracing keratin evolution: catalog, expression patterns and primary structure of shark (Scyliorhinus stellaris) keratins.

We have studied individual keratins of an elasmobranch, the shark Scyliorhinus stellaris (the lesser-spotted dogfish). From various shark tissues, notably skin and stomach, cytoskeletal proteins were isolated and then separated by two-dimensional polyacrylamide gel electrophoresis. Using complementary keratin blot-binding assays and immunoblotting, among these proteins we identified a variety of type I and type II keratins. According to their tissue-specific expression, we distinguished Is and IIs keratins from IE and IIE keratins ("S" and "E" from "simple epithelial" and "epidermal", respectively). Guinea pig antibodies which in immunoblots specifically labeled the entire range of identified shark keratins, and a monoclonal antibody specific for IE keratins were used for immunofluorescence microscopy of a broad range of shark tissues. These experiments demonstrated that in this shark, keratin expression is largely restricted to epithelia and - in contrast to the situation in teleost fishes - is lacking in mesenchymally derived cells and tissues. Peptide mass mapping of the major electrophoretically separated shark keratin spots revealed that the identified Is, IIs and IIE polypeptides are modifications of a single genuine keratin, respectively, whereas there are two different IE keratins. It, therefore, appears that in this shark most (if not all) of the keratin cytoskeleton is constituted by only five different gene products (each present in various modifications): a heterologous pair of "S" and three different "E" keratins. We sequenced three of them (Is, IIs and IIE) via cDNA cloning. Sequence alignments showed that the shark Is keratin (termed SstK18) is an ortholog of human K18, whereas the IIs keratin (termed SstK8) corresponds to human K8. In contrast, the shark IIE keratin (termed SstK1; it is the first known primary structure of a fish IIE keratin) apparently has no direct equivalent in human. On the basis of a phylogenetic tree constructed from 37 aligned keratin sequences, these results are discussed with respect to the evolution of keratin diversity in vertebrates.

Amino Acid Sequence↗
PubMed1998

Zebrafish vimentin: molecular characterization, assembly properties and developmental expression.

To provide a basis for the investigation of the intermediate filament (IF) protein vimentin in one of the most promising experimental vertebrate systems, the zebrafish (Danio rerio), we have isolated a cDNA clone of high sequence identity to and with the characteristic features of human vimentin. Using this clone we produced recombinant zebrafish vimentin and studied its assembly behaviour. Unlike other vimentins, zebrafish vimentin formed unusually thick filaments when assembled at temperatures below 21 degrees C. At 37 degrees C few filaments were observed, which often also terminated in aggregated masses, indicating that its assembly was severely disturbed at this temperature. Between 21 and 34 degrees C apparently normal IFs were generated. By viscometry, the temperature optimum of assembly was determined to be around 28 degrees C. At this temperature, zebrafish vimentin partially rescued, in mixing experiments, the temperature-dependent assembly defect of trout vimentin. Therefore it is apparently able to "instruct" the misorganized trout vimentin such that it can enter normal IFs. This feature, that assembly is best at the normal body temperature of various species, puts more weight on the assumption that vimentin is vital for some aspects of generating functional adult tissues. Remarkably, like in most other vertebrates, zebrafish vimentin appears to be an abundant factor in the lens and the retina as well as transiently, during development, in various parts of the central and peripheral nervous system. Therefore, promising cell biological investigations may now be performed with cells involved in the generation of the vertebrate eye and brain, and, in particular, the retina. Moreover, the power of genetics of the zebrafish system may be employed to investigate functional properties of vimentin in vivo.

Amino Acid Sequence↗
PubMed1997

Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH).

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 KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 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 1h, 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 KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, 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 KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.

Amino Acid Sequence↗
PubMed1997

Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 A resolution by electron cryomicroscopy and angular reconstitution.

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to approximately 45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the "functional units" within the dimeric "morphological units" of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.

Animals↗