Beta Region Research Articles

Assembly with the Na,K-ATPase α1 Subunit Is Required for Export of β1 and β2 Subunits from the Endoplasmic Reticulum

The level of the heterodimeric Na,K-ATPase is tightly controlled in epithelia to maintain appropriate transport function. The catalytic Na,K-ATPase alpha subunit is not able to exit the ER or catalyze ion transport unless assembled with the beta subunit. However, requirements for the ER exit of the Na,K-ATPase beta subunit that plays an additional, ion-transport-independent, role in intercellular adhesion are not clear. Exogenous beta(1) or beta(2) subunits expressed in renal MDCK cells replace endogenous beta(1) subunits in the alpha-beta complexes in the ER, resulting in a decrease in the amount of the alpha(1)-bound endogenous beta(1) subunits by 47-61% with no change in the amount of alpha(1) subunits. Disruption of the alpha(1)-beta association by mutations in defined alpha(1)-interacting regions of either beta(1) or beta(2) subunits results in the ER retention and rapid degradation of unassembled mutants. Hence, the ER quality control system allows export only of assembled alpha-beta complexes to the Golgi, thereby maintaining an equimolar ratio of alpha and beta subunits in the plasma membrane, whereas the number of alpha(1) subunits in the ER determines the amount of the alpha-beta complexes.

UK Myeloma Forum (UKMF) and Nordic Myeloma Study Group (NMSG): guidelines for the investigation of newly detected M‐proteins and the management of monoclonal gammopathy of undetermined significance (MGUS)

Avon Haematology Unit, Bristol Haematology and Oncology Centre, Bristol, Department of Haematology, St Helier Hospital, Carshalton, Surrey, UK, Department of Haematology, Sahlgrenska University Hospital, Gothenburg, Sweden, Department of Medicine, Malmo University Hospital, Malmo, Sweden, Division of Immunity and Infection, University of Birmingham, Birmingham, Department of Clinical Biochemistry, Frenchay Hospital, Bristol, Department Haematology, University College, London, Glasgow Western Infirmary, Glasgow, UK, Department of Haematology, NTNU/St Olavs Hospital, Trondheim, Department of Haematology, Ulleval University Hospital, Oslo, Norway, Department of Haematology Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark, and Myeloma UK.

Search for Charged Higgs Bosons in Decays of Top Quarks inpp¯Collisions ats=1.96 TeV

We report the recent charged Higgs search in top quark decays in 2.2/fb CDF data. This is the first attempt to search for charged Higgs using fully reconstructed mass assuming H->c-sbar in small tan beta region. No evidence of a charged Higgs is observed in the CDF data, hence 95% upper limits are placed at B(t->H+b) < 0.1-0.3. The upper limits can be used to limit the generic non-SM charged boson production in top quark decays.

Seric and secretory antibodies reactive to α, β and γ intimins ofEscherichia coliin healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to alpha, beta and gamma intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of alpha, beta and gamma intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.

TCR Diversity of H60-Specific CD8 T Cells During the Response Evolution and Influence of CD4 Help

H60 is a hematopoietic cell-specific dominant minor histocompatibility antigen that is considered to be ideal for modeling leukemia treatment after bone-marrow transplantation. We characterized the H60-specific CD8 T-cell response as CD4 help dependent. This study investigated the T-cell receptor (TCR) repertoires during the evolution of H60-specific CD8 T-cell responses and influence of CD4 help on the diversity. Ex vivo TCR V beta and complementarity-determining region 3 length spectratypic and clonotypic analyses were performed using H60-tetramer-binding CD8 T cells purified from the mice undergoing the primary, secondary, and tertiary responses with cognate help, and the secondary response with noncognate separate CD4 help. Involvement of a broad spectrum of TCRs was observed in the H60-specific primary response. With the involvement of diverse V beta families in the secondary and tertiary responses, complementarity-determining region 3 length and clonotypic diversities within the V beta subfamilies gradually decreased throughout the response evolution. In tertiary repertoires, the usage of V beta 8.3 and focused clonal usage within each V beta subfamily were prominent. When noncognate separate CD4 help was provided during the induction of H60-specific secondary responses, extremely limited TCRs constituted the repertoire of reactive CD8 T cells, and most of these TCRs coincided with those observed in the secondary or tertiary repertoires provided with cognate help. This study is the first to characterize the diversity of TCRs specific for hematopoietic cell-specific mouse minor H antigens and demonstrate the effect of CD4 help on CD8 TCR repertoire diversity. Our data provide a basis for modeling therapeutic applications.

The structure and functional analysis of canine T-cell receptor beta region

VDJ recombination is a key process in T-cell receptor (TCR) and immunoglobulin (Ig) molecules development. Comparison of ENSEMBL and GenBank database information revealed major differences in dog T-cell receptor beta (TRB) region annotations. ENSEMBL based genomic alignment of dog TRB sequence with human sequence and annotation showed a very similar structure of TRB. However, there is only one cluster of DJC segments in dogs. In dog, 38 V segments are followed by 1 D segment, 6 J segments and 1 C segment. Like humans and mice, dogs have another V segment opposite in orientation downstream of the C segment. V segments anticipated were analyzed using the RT-PCR and capillary electrophoresis. Thirty-one of them were identified in samples of thymus and spleen RNA and thus believed to be subjected to chromosomal rearrangement and RNA splicing. We identified and analyzed probable structure of canine TCR beta region, which is different when compared to sequences published in GenBank or ENSEMBL databases.

Zero–group-velocity modes in chalcogenide holey photonic-crystal fibers

We demonstrate that a holey photonic-crystal fiber with chalcogenide-glass index contrast can be designed to have a complete gap at a propagation constant beta = 0 that also extends into the non-zero beta region. This type of bandgap (previously identified only at index contrasts unattainable in glasses) opens up a regime for guiding zero-group-velocity modes not possible in holey fibers with the more common finger-like gaps originating from beta-->infinity. Such modes could be used to enhance nonlinear and other material interactions, such as for hollow-core fibers in gas-sensor applications.

Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.

Two-stage genome-wide association study identifies integrin beta 5 as having potential role in bull fertility

BackgroundFertility is one of the most critical factors controlling biological and financial performance of animal production systems and genetic improvement of lines. The objective of this study was to identify molecular defects in the sperm that are responsible for uncompensable fertility in Holstein bulls. We performed a comprehensive genome wide analysis of single nucleotide polymorphisms (SNP) for bull fertility followed by a second-stage replication in additional bulls for a restricted set of markers.ResultsIn the Phase I association study, we genotyped the genomic sperm DNA of 10 low-fertility and 10 high-fertility bulls using Bovine SNP Gene Chips containing approximately 10,000 random SNP markers. In these animals, 8,207 markers were found to be polymorphic, 97 of which were significantly associated with fertility (p < 0.01). In the Phase II study, we tested the four most significant SNP from the Phase I study in 101 low-fertility and 100 high-fertility bulls, with two SNPs (rs29024867 and rs41257187) significantly replicated. Rs29024867 corresponds to a nucleotide change of C → G 2,190 bp 3' of the collagen type I alpha 2 gene on chromosome 4, while the rs41257187 (C → T) is in the coding region of integrin beta 5 gene on chromosome 1. The SNP rs41257187 induces a synonymous (Proline → Proline), suggesting disequilibrium with the true causative locus (i), but we found that the incubation of bull spermatozoa with integrin beta 5 antibodies significantly decreased the ability to fertilize oocytes. Our findings suggest that the bovine sperm integrin beta 5 protein plays a role during fertilization and could serve as a positional or functional marker of bull fertility.ConclusionWe have identified molecular markers associated with bull fertility and established that at least one of the genes harboring such variation has a role in fertility. The findings are important in understanding mechanisms of uncompensatory infertility in bulls, and in other male mammals. The findings set the stage for more hypothesis-driven research aimed at discovering the role of variation in the genome that affect fertility and that can be used to identify molecular mechanisms of development.

Specific mutations in the enhancer II/core promoter/precore regions of hepatitis B virus subgenotype C2 in Korean patients with hepatocellular carcinoma

Recently, hepatitis B virus (HBV) genotypes and mutations have been reported to be related to hepatocellular carcinoma (HCC). This cross-sectional case-control study examined the relationship between HCC and mutations in the enhancer II/core promoter and precore regions of HBV by comparing 135 Korean HCC patients infected with HBV genotype C2 (HBV/C2; HCC group) with 135 age-, sex-, and hepatitis B e antigen (HBeAg) status-matched patients without HCC (non- HCC group). Age and sex were also matched between HBeAg-positive and -negative patients. The prevalence of T1653, A1689, V1753, T1762/A1764, T1846, A1850, C1858, and A1896 mutations was evaluated in this population. The prevalence of the T1653 mutation in the box alpha region, the T1689 [corrected] mutation in between the box alpha and beta regions, and the T1762/A1764 mutations in the basal core promoter region was significantly higher in the HCC group compared to the non-HCC group (8.9% vs. 2.2%, P = 0.017; 19.3% vs. 4.4%, P < 0.001; and 60.7% vs. 22.2%; P < 0.001). Among HBeAg-negative patients, the frequency of the T1653 mutation was higher in the HCC group. Regardless of HBeAg status, the prevalence of the T1689, [corrected] and T1762/A1764 mutations was higher in the HCC group than in the non-HCC group. However, no association was observed between mutations in the precore region and HCC. Upon multivariate analysis, the presence of the T1653, T1689, [corrected] and T1762/A1764 mutations was an independent predictive factor for HCC. The addition of the T1653 or T1689 [corrected] mutation to T1762/A1764 increased the risk of HCC. In conclusion, the T1653, T1689, [corrected] and/or T1762/A1764 mutations were associated with the development of HCC in Korean patients infected with HBV/C2.

Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA

The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).

Structural determinants of monohydroxylated bile acids to activate β1 subunit-containing BK channels

Lithocholate (LC) (10-300 microM) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel beta(1) accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1+beta(1) subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5beta-cholanic acid-3alpha-ol), 5alpha-cholanic acid-3alpha-ol, and 5beta-cholanic acid-3beta-ol increased NPo (EC(50) approximately 45 microM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol raised NPo by 40%. Thus, the alpha-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol partially matched LC shape, and 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol did not. Increasing polarity in steroid rings (5beta-cholanic acid-3alpha-sulfate) or reducing polarity in lateral chain (5beta-cholanic acid 3alpha-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between beta(1) and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in beta(1) transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.

The I260Q Variant of DNA Polymerase β Extends Mispaired Primer Termini Due to Its Increased Affinity for Deoxynucleotide Triphosphate Substrates

DNA polymerase beta plays a key role in base excision repair. We have previously shown that the hydrophobic hinge region of polymerase beta, which is distant from its active site, plays a critical role in the fidelity of DNA synthesis by this enzyme. The I260Q hinge variant of polymerase beta misincorporates nucleotides with a significantly higher catalytic efficiency than the wild-type enzyme. In the study described here, we show that I260Q extends mispaired primer termini. The kinetic basis for extension of mispairs is defective discrimination by I260Q at the level of ground-state binding of the dNTP substrate. Our results suggest that the hydrophobic hinge region influences the geometry of the dNTP binding pocket exclusively. Because the DNA forms part of the binding pocket, our data are also consistent with the interpretation that the mispaired primer terminus affects the geometry of the dNTP binding pocket such that the I260Q variant has a higher affinity for the incoming dNTP than wild-type polymerase beta.

Molecular Dynamics Simulations To Investigate the Domain Swapping Mechanism of Human Cystatin C

Human cystatin C (HCC), one of the amyloidgenic proteins, has been proved to form a dimeric structure via a domain swapping process and then cause amyloid deposits in the brains of patients suffering from Alzheimer's disease. HCC monomer consists of a core with a five-stranded antiparallel beta-sheet (beta region) wrapped around a central helix. The connectivity of these secondary structures is: (N)-beta1-alpha-beta2-L1-beta3-AS-beta4-L2-beta5-(C). In this study, various molecular dynamics simulations were conducted to investigate the conformational changes of the monomeric HCC at different temperatures (300 and 500 K) and pH levels (2, 4, and 7) to gain insight into the domain swapping mechanism. The results show that high temperature (500 K) and low pH (pH 2) will trigger the domain swapping process of HCC. We further proposed that the domain swapping mechanism of HCC follows four steps: (1) the alpha-helix moves away from the beta region; (2) the contacts between beta2 and beta3-AS disappear; (3) the beta2-L1-beta3 hairpin unfolds following the so-called "zip-up" mechanism; and finally (4) the HCC dimer is formed. Our study shows that high temperature can accelerate the unfolding of HCC and the departure of the alpha-helix from the beta-region, especially at low pH value. This is attributed to the fact that that low pH results in the protonation of the side chains of Asp, Glu, and His residues, which further disrupts the following four salt-bridge interactions stabilizing the alpha-beta interface of the native structure: Asp15-Arg53 (beta1-beta2), Glu21/20-Lys54 (helix-beta2), Asp40-Arg70 (helix-AS), and His43-Asp81 (beta2-AS).

Integrin β3 expression is regulated by let-7a miRNA in malignant melanoma

Although integrin beta(3) is known to play an important role in melanoma progression and invasion, regulation of integrin beta(3) expression in melanoma has not been analysed in detail until today. As transcriptional regulation of integrin beta(3) was ruled out by our analysis, we concentrated on the regulation by microRNAs (miRNAs). Comparing primary melanocytes and malignant melanoma cell lines, we found that one candidate miRNA, miR-let-7a, was lost in melanoma and sequence analysis suggested an interaction with the 3'-untranslated region (3'-UTR) of integrin beta(3) mRNA. Transfection of melanoma cells with let-7a pre-miR(TM) molecules resulted in the downregulation of integrin beta(3) mRNA and protein expression. In addition, we cloned the 3'-UTR of the integrin beta(3) mRNA containing the let-7a target sequence into a reporter plasmid and revealed that let-7a negatively regulates reporter gene expression. The repressed expression of integrin beta(3) accompanies with reduced invasive potential of melanoma cells transfected with synthetic let-7a molecules observed in Boyden chamber assays. On the other hand, the induction of integrin beta(3) expression was achieved in melanocytes by transfection with let-7a anti-miRs, resulting in invasive behavior of transfected melanocytes. In summary, we determined miRNA let-7a to be an important regulator of integrin beta(3) expression and showed that the loss of let-7a expression is involved in development and progression of malignant melanoma.

A coupled EBSD/EDS method to determine the primary- and secondary-alpha textures in titanium alloys with duplex microstructures

A method for separating the textures of primary alpha (αp) and secondary alpha (αs) in alpha/beta titanium alloys with a duplex microstructure was developed. Utilizing electron backscatter diffraction (EBSD) and energy-dispersive spectroscopy (EDS), the approach relies on the non-uniform partitioning of alloying elements between primary alpha and regions containing secondary-alpha lamellae and residual beta matrix phase. The method was evaluated using samples of Ti–6Al–4V for which vanadium partitions strongly to secondary alpha+beta regions. The technique thus provides a useful tool for quantifying the evolution of deformation texture in the primary alpha and transformation texture in secondary alpha formed via decomposition of the beta matrix following hot working or final heat treatment.

Comprehensive analysis of the helix‐X‐helix motif in soluble proteins

Alpha-helices are the most common secondary structures found in globular proteins. In this report, we analyze the stereochemical and sequence properties of helix-X-helix (HXH) motifs in which two alpha-helices are linked by a single residue, in search of characteristic structures and sequence signals. The analysis is carried out on a database of 837 nonredundant HXH motifs. The kinks are characterized by the bend angle between the axes of the N-terminal and C-terminal helices and the wobble angle corresponding to the rotation of C-terminal helix axis on the plane perpendicular to the N-terminal one. The phi-psi dihedral angles of the linker residue are clustered in six distinct areas of the Ramachandran plot: two areas are located in the additional allowed alpha region (alpha(1) and alpha(2)), two areas are in the additional allowed beta region (beta(1) and beta(2)) and two areas have positive phi values (alpha(L) and beta(M)). Each phi/psi region corresponds to characteristic bend and wobble angles and amino acid distributions. Bend angles can vary from 0 degrees to 160 degrees. Most wobble angles correspond to a counter-clockwise rotation of the C-terminal helix. Proline residues are rigorously excluded from the linker position X but have a high propensity at position X+1 of the beta(1) and beta(2) motifs (12 and 7, respectively) and at position X+3 of the alpha(1) motifs (9). Glycine linkers are located either in the alpha(L) region (20%) or in the beta(M) region (80%). This latter conformation is characterized by a marked bend angle (124 degrees +/- 18 degrees) and a clockwise wobble. Among other amino acids, Asn is remarkable for its high propensity (>3) at the linker position of the alpha(2), beta(1), and beta(2) motifs. Stabilization of HXH motifs by H-bonds between polar side chains of the linker and polar groups of the backbone is determined. A method based on position-specific scoring matrices is developed for conformational prediction. The accuracy of the predictions reaches 80% when the method is applied to proline-induced kinks or to kinks with bend angles in the 50 degrees-100 degrees range.

Comparison between a second generation automated multicapillary electrophoresis system with an automated agarose gel electrophoresis system for the detection of M-components

During the last decade, capillary electrophoresis (CE) has emerged as an interesting alternative to traditional analysis of serum, plasma and urine proteins by agarose gel electrophoresis. Initially there was a considerable difference in resolution between the two methods but the quality of CE has improved significantly. We thus wanted to evaluate a second generation of automated multicapillary instruments (Capillarys, Sebia, Paris, France) and the high resolution (HR) buffer for serum or plasma protein analysis with an automated agarose gel electrophoresis system for the detection of M-components. The comparison between the two systems was performed with patients samples with and without M-components. The comparison included 76 serum samples with M-components > 1 g/L. There was a total agreement between the two methods for detection of these M-components. When studying samples containing oligoclonal bands/small M-components, there were differences between the two systems. The capillary electrophoresis system detected a slightly higher number of samples with oligoclonal bands but the two systems found oligoclonal bands in different samples. When looking at resolution, the agarose gel electrophoresis system yielded a slightly better resolution in the alpha and beta regions, but it required an experienced interpreter to be able to benefit from the increased resolution. The capillary electrophoresis has shorter turn-around times and bar-code reader that allows positive sample identification. The Capillarys in combination with HR buffer gives better resolution of the alpha and beta regions than the same instrument with the ß1-ß2+ buffer or the Paragon CZE2000® (Beckman) which was the first generation of capillary electrophoresis systems.

Monoclonal Immunoglobulins of Alpha-2 Mobility on Protein Electrophoresis: A Case Series.

Protein electrophoresis (PEP) separates proteins based on mobility through support media such as agarose into five general regions: albumin, alpha-1, alpha-2, beta, and gamma. Monoclonal and polyclonal immunoglobulins typically migrate in the gamma or beta regions. Between April 1990 and July 2007 at Columbia University Medical Center, we identified and then retrospectively reviewed 15 patients with monoclonal proteins of alpha-2 mobility repeatedly on serum PEP (see figure). Immunofixation identified the paraprotein as IgA kappa in 7 patients, IgA lambda in 2, IgG lambda in 2, free kappa light chain in 4, free lambda light chain in 2, and one IgG lambda. In the urine, of the 14 patients who had PEP performed, 6 patients had serum concordant light chains (5 kappa and 1 lambda) also migrating in the alpha 2 region, however, for 5 other patients migration was either in the beta or gamma regions. Published case reports of monoclonal proteins with atypical mobility patterns are reviewed. The median age of our patients was 66 years and there were 9 males. 10 patients had multiple myeloma, 5 had monoclonal gammopathy of undetermined significance (MGUS), and 1 had lambda AL amyloidosis. This is the largest case series ever described of patients with monoclonal proteins migrating in alpha 2 region and illustrates the importance of immunofixation in screening patients for plasma cell dyscrasias. Possible explanations for this unusual migration pattern include protein complex formation with other plasma components, high carbohydrate content, or given the IgA predominance noted, properties of the IgA immunoglobulin such as polymerization. [Display omitted]

Structural features and functional domains of amassin-1, a cell-binding olfactomedin protein

Amassin-1 mediates a rapid cell adhesion that tightly adheres sea urchin coelomocytes (body cavity immunocytes) together. Three major structural regions exist in amassin-1: a short beta region, 3 coiled coils, and an olfactomedin domain. Amassin-1 contains 8 disulfide-bonded cysteines that, upon reduction, render it inactive. Truncated forms of recombinant amassin-1 were expressed and purified from Pichia pastoris and their disulfide bonding and biological activities investigated. Expressed alone, the olfactomedin domain contained 2 intramolecular disulfide bonds, existed in a monomeric state, and inhibited amassin-1-mediated clotting of coelomocytes by a calcium-dependent cell-binding activity. The N-terminal beta region, containing 3 cysteines, was not required for clotting activity. The coiled coils may dimerize amassin-1 in a parallel orientation through a homodimerizing disulfide bond. Neither amassin-1 fragments that were disulfide-linked as dimers or that were engineered to exist as dimers induced coelomocytes clotting. Clotting required higher multimeric states of amassin-1, possibly tetramers, which occurred through the N-terminal beta region and (or) the first segment of coiled coils.