Eukaryotic Initiation Factor 2B (eIF2B) GEF Activity as a Diagnostic Tool for EIF2B-Related Disorders

Background

In recent years, the phenotypes of leukodystrophies linked to mutations in the eukaryotic initiation factor 2B genes have been extended, classically called CACH/VWM (Childhood ataxia with cntral hypomyélination/vanishing white matter disorder). The large clinical spectrum observed from the more severe antenatal forms responsible for fetal death to milder adult forms with an onset after 16 years old and restricted to slow cognitive impairment have lead to the concept of eIF2B-related disorders. The typical MRI pattern with a diffuse CSF-like aspect of the cerebral white matter can lack particularly in the adult forms whereas an increasing number of patients with clinical and MRI criteria for CACH/VWM disease but without eIF2B mutations are found. Then we propose the use of biochemical markers to help in this difficult diagnosis. The biochemical diagnosis of eIF2B-related disorder is difficult as no marker, except the recently described asialotransferrin/transferrin ratio measured in cerebrospinal fluid, has been proposed and validated until now. Decreased eIF2B GEF activity has been previously reported in lymphoblastoid cell lines from 30 eIF2B-mutated patients. Our objective was to evaluate further the utility of this marker and to validate eIF2B GEF activity in a larger cohort as a specific diagnostic test for eIF2B-related disorders.

Methodology/Principal Findings

We performed eIF2B GEF activity assays in cells from 63 patients presenting with different clinical forms and eIF2B mutations in comparison to controls but also to patients with defined leukodystrophies or CACH/VWM-like diseases without eIF2B mutations. We found a significant decrease of GEF activity in cells from eIF2B-mutated patients with 100% specificity and 89% sensitivity when the activity threshold was set at ≤77.5%.

Conclusion

These results validate the measurement of eIF2B GEF activity in patients'' transformed-lymphocytes as an important tool for the diagnosis of eIF2B-related disorders.     

Crystal Structure of the α Subunit of Human Translation Initiation Factor 2B

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the α subunit of human eIF2B at 2.65 Å resolution. The eIF2Bα structure consists of the N-terminal α-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 Å in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 Å in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Bα. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Bα. Interestingly, the yeast eIF2Bα missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Bα missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The β and δ subunits of eIF2B are homologous to eIF2Bα and may have tertiary structures similar to the present eIF2Bα structure. The sulfate-ion-binding residues of eIF2Bα are well conserved in eIF2Bβ/δ. The abovementioned yeast and human missense mutations of eIF2Bβ/δ were also mapped on the eIF2Bα structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.     

Mutations causing childhood ataxia with central nervous system hypomyelination reduce eukaryotic initiation factor 2B complex formation and activity

Childhood ataxia with central nervous system hypomyelination (CACH), or vanishing white matter leukoencephalopathy (VWM), is a fatal brain disorder caused by mutations in eukaryotic initiation factor 2B (eIF2B). eIF2B is essential for protein synthesis and regulates translation in response to cellular stresses. We performed mutagenesis to introduce changes equivalent to 12 human CACH/VWM mutations in three subunits of the equivalent factor from yeast (Saccharomyces cerevisiae) and analyzed effects on cell growth, translation, and gene expression in response to stresses. None of the mutations is lethal or temperature sensitive, but almost all confer some defect in eIF2B function significant enough to alter growth or gene expression under normal or stress conditions. Biochemical analyses indicate that mutations analyzed in eIF2Balpha and -epsilon reduce the steady-state level of the affected subunit, while the most severe mutant tested, eIF2Bbeta(V341D) (human eIF2B(betaV316D)), forms complexes with reduced stability and lower eIF2B activity. eIF2Bdelta is excluded from eIF2Bbeta(V341D) complexes. eIF2B(betav341D) function can be rescued by overexpression of eIF2Bdelta alone. Our findings imply CACH/VWM mutations do not specifically impair responses to eIF2 phosphorylation, but instead cause protein structure defects that impair eIF2B activity. Altered protein folding is characteristic of other diseases, including cystic fibrosis and neurodegenerative disorders such as Huntington, Alzheimer's, and prion diseases.     

Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.     

Mutations linked to leukoencephalopathy with vanishing white matter impair the function of the eukaryotic initiation factor 2B complex in diverse ways

Leukoencephalopathy with vanishing white matter (VWM) is a severe inherited human neurodegenerative disorder that is caused by mutations in the genes for the subunits of eukaryotic initiation factor 2B (eIF2B), a heteropentameric guanine nucleotide exchange factor that regulates both global and mRNA-specific translation. Marked variability is evident in the clinical severity and time course of VWM in patients. Here we have studied the effects of VWM mutations on the function of human eIF2B. All the mutations tested cause partial loss of activity. Frameshift mutations in genes for eIF2Bepsilon or eIF2Bbeta lead to truncated polypeptides that fail to form complexes with the other subunits and are effectively null mutations. Certain point mutations also impair the ability of eIF2Bbeta or -epsilon to form eIF2B holocomplexes and also diminish the intrinsic nucleotide exchange activity of eIF2B. A point mutation in the catalytic domain of eIF2Bepsilon impairs its ability to bind the substrate, while two mutations in eIF2Bbeta actually enhance eIF2 binding. We provide evidence that expression of VWM mutant eIF2B may enhance the translation of specific mRNAs. The variability of the clinical phenotype in VWM may reflect the multiple ways in which VWM mutations affect eIF2B function.     

Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bepsilon). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bepsilon in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2Bepsilon (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bepsilon (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bepsilon without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bepsilon disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bepsilon alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bepsilon alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bepsilon and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bepsilon is an activation domain that responds to eIF2B complex formation.     

CSF N-Glycan Profiles to Investigate Biomarkers in Brain Developmental Disorders: Application to Leukodystrophies Related to eIF2B Mutations

Background

Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker.

Methodology/Principal Findings

We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders.

Conclusion

Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.     

Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2.

eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit [eIF2(alphaP)]. Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.     

A Point Mutation in Translation Initiation Factor 2B Leads to a Continuous Hyper Stress State in Oligodendroglial-Derived Cells

Background

Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM). The disease is manifested by loss of brain myelin upon physiological stress. In a previous study, we showed that fibroblasts isolated from CACH/VWM patients are hypersensitive to pharmacologically-induced endoplasmic reticulum (ER) stress. Since brain cells from affected individuals are not available for research, we wished to assess the effect of eIF2B mutation on oligodendroglial-derived cells.

Methodology/Principal Findings

A rat oligodendroglial-derived cell line was used for a stable knock-down of eIF2B5 followed by stable expression of mutated eIF2B5(R195H) cDNA. In response to a pharmacological ER-stress agent, eIF2B5(R195H) expressing cells exhibited heightened ER-stress response demonstrated by hyper induction of ATF4, GADD34, Bip, PDIA1, PDIA3, PDIA4 and PDIA6 proteins. Moreover, even in the absence of a pharmacological stress agent, eIF2B5(R195H)-expressing cells exhibited high basal levels of ATF4, GADD34 and ER-associated Bip, PDIA1 and PDIA3.

Significance

The data provide evidence that oligodendroglial-derived cells expressing a mutated eIF2B constantly use their stress response mechanism as an adaptation mean in order to survive. The current study is the first to demonstrate the effects of eIF2B5 mutation on ER homeostasis in oligodendroglial-derived cells.     

Transgenic Brassica rapa plants over‐expressing eIF(iso)4E variants show broad‐spectrum Turnip mosaic virus (TuMV) resistance

The protein–protein interaction between VPg (viral protein genome‐linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad‐spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge‐based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap‐binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap‐binding pockets, and mutated. Yeast two‐hybrid assay and co‐immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E‐knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild‐type were over‐expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T₁ and T₂ transformants demonstrated that the over‐expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge‐based approaches for the engineering of broad‐spectrum resistance in Chinese cabbage.     

eIF2B-related disorders: antenatal onset and involvement of multiple organs

Leukoencephalopathy with vanishing white matter, also called "childhood ataxia with central nervous system hypomyelination," is the first human disease related to mutations in any of the five genes encoding subunits of eukaryotic initiation factor eIF2B or any translation factor at all. eIF2B is essential in all cells of the body for protein synthesis and the regulation of this protein synthesis under different stress conditions. It is surprising that mutations in the eIF2B genes have been reported to lead to abnormalities of the white matter of the brain only, although it has been shown recently that ovarian failure may accompany the leukoencephalopathy. Another surprising observation is that the onset of the disease varies from early childhood to adulthood, with the exception of Cree leukoencephalopathy, a disease related to a particular mutation in one of the eIF2B genes, which invariably has its onset within the first year of life. We analyzed the eIF2B genes of nine patients with an antenatal- or early-infantile-onset encephalopathy and an early demise and found mutations in eight of the patients. In addition to signs of a serious encephalopathy, we found oligohydramnios, intrauterine growth retardation, cataracts, pancreatitis, hepatosplenomegaly, hypoplasia of the kidneys, and ovarian dysgenesis. Until now, no evidence had been found for a genotype-phenotype correlation, but the consistently severe phenotype in affected siblings among our patients and in Cree encephalopathy patients suggests an influence of the genotype on the phenotype.     

A new function and complexity for protein translation initiation factor eIF2B

eIF2B is a multisubunit protein that is critical for protein synthesis initiation and its control. It is a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2. eIF2 binds initiator tRNA to ribosomes and promotes mRNA AUG codon recognition. eIF2B is critical for regulation of protein synthesis via a conserved mechanism of phosphorylation of eIF2, which converts eIF2 from a substrate to an inhibitor of eIF2B GEF. In addition, inherited mutations affecting eIF2B subunits cause the fatal disorder leukoencephalopathy with Vanishing White Matter (VWM), also called Childhood Ataxia with Central nervous system Hypomyelination (CACH). Here we review findings which reveal that eIF2B is a decameric protein and also define a new function for the eIF2B. Our results demonstrate that the eIF2Bγ subunit is required for eIF2B to gain access to eIF2•GDP. Specifically it displaces a third translation factor eIF5 (a dual function GAP and GDI) from eIF2•GDP/eIF5 complexes. Thus eIF2B is a GDI displacement factor (or GDF) in addition to its role as a GEF, prompting the redrawing of the eIF2 cycling pathway to incorporate the new steps. In structural studies using mass spectrometry and cross-linking it is shown that eIF2B is a dimer of pentamers and so is twice as large as previously thought. A binding site for GTP on eIF2B was also found, raising further questions concerning the mechanism of nucleotide exchange. The implications of these findings for eIF2B function and for VWM/CACH disease are discussed.     

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2α kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.     

Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.     

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.     

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

In eukaryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2.GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bvarepsilon catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bvarepsilon mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bvarepsilon with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bvarepsilon segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bvarepsilon to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA(i)(Met), suggesting that tRNA(i)(Met) is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.     

Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc-3 is associated with the homozygotic presence of a mutated eIF4E allele

Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris , four recessive genes, bc-1 , bc-2 , bc-3 and bc-u , have been proposed to control resistance to the potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus . In order to identify molecular entities for these genes, we cloned and sequenced P. vulgaris homologues of genes encoding the eIF proteins eIF4E, eIF(iso)4E and nCBP. Bean genotypes reported to carry bc-3 resistance were found specifically to carry non-silent mutations at codons 53, 65, 76 and 111 in eIF4E . This set of mutations closely resembled a pattern of eIF4E mutations determining potyvirus resistance in other plant species. The segregation of BCMV resistance and eIF4E genotype was subsequently analysed in an F₂ population derived from the P. vulgaris all-susceptible genotype and a genotype carrying bc-3 . F₂ plants homozygous for the eIF4E mutant allele were found to display at least the same level of resistance to BCMV as the parental resistant genotype. At 6 weeks after inoculation, all F₂ plants found to be BCMV negative by enzyme-linked immunosorbent assay were found to be homozygous for the mutant eIF4E allele. In F₃ plants homozygous for the mutated allele, virus resistance was subsequently found to be stably maintained. In conclusion, allelic eIF4E appears to be associated with a major component of potyvirus resistance present in bc-3 genotypes of bean.