Insertions in the OCL1 locus of Acinetobacter baumannii lead to shortened lipooligosaccharides

Genomes of 82 Acinetobacter baumannii global clones 1 (GC1) and 2 (GC2) isolates were sequenced and different forms of the locus predicted to direct synthesis of the outer core (OC) of the lipooligosaccharide were identified. OCL1 was in all GC2 genomes, whereas GC1 isolates carried OCL1, OCL3 or a new locus, OCL5. Three mutants in which an insertion sequence (ISAba1 or ISAba23) interrupted OCL1 were identified. Isolates with OCL1 intact produced only lipooligosaccharide, while the mutants produced lipooligosaccharide of reduced molecular weight. Thus, the assignment of the OC locus as that responsible for the synthesis of the OC is correct.

Variation in the OC locus of Acinetobacter baumannii genomes predicts extensive structural diversity in the Lipooligosaccharide

Lipooligosaccharide (LOS) is a complex surface structure that is linked to many pathogenic properties of Acinetobacter baumannii. In A. baumannii, the genes responsible for the synthesis of the outer core (OC) component of the LOS are located between ilvE and aspS. The content of the OC locus is usually variable within a species, and examination of 6 complete and 227 draft A. baumannii genome sequences available in GenBank non-redundant and Whole Genome Shotgun databases revealed nine distinct new types, OCL4-OCL12, in addition to the three known ones. The twelve gene clusters fell into two distinct groups, designated Group A and Group B, based on similarities in the genes present. OCL6 (Group B) was unique in that it included genes for the synthesis of L-Rhamnosep. Genetic exchange of the different configurations between strains has occurred as some OC forms were found in several different sequence types (STs). OCL1 (Group A) was the most widely distributed being present in 18 STs, and OCL6 was found in 16 STs. Variation within clones was also observed, with more than one OC locus type found in the two globally disseminated clones, GC1 and GC2, that include the majority of multiply antibiotic resistant isolates. OCL1 was the most abundant gene cluster in both GC1 and GC2 genomes but GC1 isolates also carried OCL2, OCL3 or OCL5, and OCL3 was also present in GC2. As replacement of the OC locus in the major global clones indicates the presence of sub-lineages, a PCR typing scheme was developed to rapidly distinguish Group A and Group B types, and to distinguish the specific forms found in GC1 and GC2 isolates.

The MS risk allele of CD40 is associated with reduced cell-membrane bound expression in antigen presenting cells: Implications for gene function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. © 2015 Field et al.

Polymorphisms in the receptor tyrosine kinase MERTK gene are associated with Multiple Sclerosis susceptibility

Multiple sclerosis (MS) is a debilitating, chronic demyelinating disease of the central nervous system affecting over 2 million people worldwide. The TAM family of receptor tyrosine kinases (TYRO3, AXL and MERTK) have been implicated as important players during demyelination in both animal models of MS and in the human disease. We therefore conducted an association study to identify single nucleotide polymorphisms (SNPs) within genes encoding the TAM receptors and their ligands associated with MS. Analysis of genotype data from a genome-wide association study which consisted of 1618 MS cases and 3413 healthy controls conducted by the Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene) revealed several SNPs within the MERTK gene (Chromosome 2q14.1, Accession Number NG_011607.1) that showed suggestive association with MS. We therefore interrogated 28 SNPs in MERTK in an independent replication cohort of 1140 MS cases and 1140 healthy controls. We found 12 SNPs that replicated, with 7 SNPs showing p-values of less than 10-5 when the discovery and replication cohorts were combined. All 12 replicated SNPs were in strong linkage disequilibrium with each other. In combination, these data suggest the MERTK gene is a novel risk gene for MS susceptibility. © 2011 Ma et al.

Whole blood transcriptional profiling in ankylosing spondylitis identifies novel candidate genes that might contribute to the inflammatory and tissue-destructive disease aspects

Introduction: A number of genetic-association studies have identified genes contributing to ankylosing spondylitis (AS) susceptibility but such approaches provide little information as to the gene activity changes occurring during the disease process. Transcriptional profiling generates a 'snapshot' of the sampled cells' activity and thus can provide insights into the molecular processes driving the disease process. We undertook a whole-genome microarray approach to identify candidate genes associated with AS and validated these gene-expression changes in a larger sample cohort. Methods: A total of 18 active AS patients, classified according to the New York criteria, and 18 gender- and age-matched controls were profiled using Illumina HT-12 whole-genome expression BeadChips which carry cDNAs for 48,000 genes and transcripts. Class comparison analysis identified a number of differentially expressed candidate genes. These candidate genes were then validated in a larger cohort using qPCR-based TaqMan low density arrays (TLDAs). Results: A total of 239 probes corresponding to 221 genes were identified as being significantly different between patients and controls with a P-value <0.0005 (80% confidence level of false discovery rate). Forty-seven genes were then selected for validation studies, using the TLDAs. Thirteen of these genes were validated in the second patient cohort with 12 downregulated 1.3- to 2-fold and only 1 upregulated (1.6-fold). Among a number of identified genes with well-documented inflammatory roles we also validated genes that might be of great interest to the understanding of AS progression such as SPOCK2 (osteonectin) and EP300, which modulate cartilage and bone metabolism. Conclusions: We have validated a gene expression signature for AS from whole blood and identified strong candidate genes that may play roles in both the inflammatory and joint destruction aspects of the disease.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

VH gene usage in immunoglobulin E responses of seasonal rhinitis patients allergic to grass pollen is oligoclonal and antigen driven

Background: IgE is the pivotal-specific effector molecule of allergic reactions yet it remains unclear whether the elevated production of IgE in atopic individuals is due to superantigen activation of B cell populations, increased antibody class switching to IgE or oligoclonal allergen-driven IgE responses. Objectives: To increase our understanding of the mechanisms driving IgE responses in allergic disease we examined immunoglobulin variable regions of IgE heavy chain transcripts from three patients with seasonal rhinitis due to grass pollen allergy. Methods: Variable domain of heavy chain-epsilon constant domain 1 cDNAs were amplified from peripheral blood using a two-step semi-nested PCR, cloned and sequenced. Results: The VH gene family usage in subject A was broadly based, but there were two clusters of sequences using genes VH 3-9 and 3-11 with unusually low levels of somatic mutations, 0-3%. Subject B repeatedly used VH 1-69 and subject C repeatedly used VH 1-02, 1-46 and 5a genes. Most clones were highly mutated being only 86-95% homologous to their germline VH gene counterparts and somatic mutations were more abundant at the complementarity determining rather than framework regions. Multiple sequence alignment revealed both repeated use of particular VH genes as well as clonal relatedness among clusters of IgE transcripts. Conclusion: In contrast to previous studies we observed no preferred VH gene common to IgE transcripts of the three subjects allergic to grass pollen. Moreover, most of the VH gene characteristics of the IgE transcripts were consistent with oligoclonal antigen-driven IgE responses.

A comparative study of antibody expressions in primary biliary cirrhosis and autoimmune cholangitis using phage display

Primary biliary cirrhosis (PBC) and autoimmune cholangitis (AIC) are serologic expressions of an autoimmune liver disease affecting biliary ductular cells. Previously we screened a phage-displayed random peptide library with polyclonal IgG from 2 Australian patients with PBC and derived peptides that identified a single conformational (discontinuous) epitope in the inner lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the characteristic autoantigen in PBC. Here we have used phage display to investigate the reactivity of PBC sera from 2 ethnically and geographically distinct populations, Japanese and Australian, and the 2 serologic expressions, PBC and AIC. Random 7-mer and 12-mer peptide libraries were biopanned with IgG from 3 Japanese patients with PBC and 3 with AIC who did not have anti-PDC-E2. The phage clones (phagotopes) obtained were tested by capture enzyme-linked immunosorbent assay (ELISA) for reactivity with affinity-purified anti-PDC-E2, and compared with those obtained from Australian patients with PBC. Peptide sequences of the derived phagotopes and sequences derived by biopanning with irrelevant antisera were aligned to develop a guide tree based on physicochemical similarity. Both Australian and Japanese PBC-derived phagotopes were distributed in branches of the guide tree that contained the peptide sequences MH and FV previously identified as part of an immunodominant conformational epitope of PDC-E2, indicating that epitope selection was not influenced by the racial origin of the PBC sera. Biopanning with either PBC or AIC-derived IgG yielded phagotopes that reacted with anti-PDC-E2 by capture ELISA, further establishing that there is a similar autoimmune targeting in PBC and AIC.

Towards the pathogenesis of autoimmune liver disease

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Association of IL23R and ERAP1 genes with ankylosing spondylitis in a Portuguese population

Objective: Association between ankylosing spondylitis (AS) and two genes, ERAP1 and IL23R, has recently been reported in North American and British populations. The population attributable risk fraction for ERAP1 in this study was 25%, and for IL23R, 9%. Confirmation of these findings to ERAP1 in other ethnic groups has not yet been demonstrated. We sought to test the association between single nucleotide polymorphisms (SNPs) in these genes and susceptibility to AS among a Portuguese population. We also investigated the role of these genes in clinical manifestations of AS, including age of symptom onset, the Bath Ankylosing Spondylitis Disease Activity, Metrology and Functional Indices, and the modified Stoke Ankylosing Spondylitis Spinal Score. Methods: The study was conducted on 358 AS cases and 285 ethnically matched Portuguese healthy controls. AS was defined according to the modified New York Criteria. Genotyping of IL23R and ERAP1 allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests of genotypes as implemented in PLINK for dichotomous and quantitative variables respectively. A meta-analysis for Portuguese and previously published Spanish IL23R data was performed using the StatsDirect® Statistical tools, by fixed and random effects models. Results: A total of 14 nsSNPs markers (8 for IL23R, 5 for ERAPl, 1 for LN-PEP) were analysed. Three markers (2 for IL23R and 1 for ERAP1) showed significant single-locus disease associations, confirming that the association of these genes with AS in the Portuguese population. The strongest associated SNP in IL23R was rs1004819 (OR=1.4, p=0.0049), and in ERAP1 was rs30187 (OR=1.26, p=0.035). The population attributable risk fractions in the Portuguese population for these SNPs are 11% and 9.7% respectively. No association was seen with any SNP in LN-PEP, which flanks ERAP1 and was associated with AS in the British population. No association was seen with clinical manifestations of AS. Conclusions: These results show that IL23R and ERAP1 genes are also associated with susceptibility to AS in the Portuguese population, and that they contribute a significant proportion of the population risk for this disease.

Spectrum of ankylosing spondylitis in Portugal. Development of BASDAI, BASFI, BASMI and mSASSS reference centile charts

The availability of population-specific normative data regarding disease severity measures is essential for patient assessment. The goals of the current study were to characterize the pattern of ankylosing spondylitis (AS) in Portuguese patients and to develop reference centile charts for BASDAI, BASFI, BASMI and mSASSS, the most widely used assessment tools in AS. AS cases were recruited from hospital outpatient clinics, with AS defined according to the modified New York criteria. Demographic and clinical data were recorded. All radiographs were evaluated by two independent experienced readers. Centile charts for BASDAI, BASFI, BASMI and mSASSS were constructed for both genders, using generalized linear models and regression models with duration of disease as independent variable. A total of 369 patients (62.3% male, mean ± (SD) age 45.4 ± 13.2 years, mean ± (SD) disease duration 11.4 ± 10.5 years, 70.7% B27-positive) were included. Family history of AS in a first-degree relative was reported in 17.6% of the cases. Regarding clinical disease pattern, at the time of assessment 42.3% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopatic disease. Anterior uveitis (33.6%) was the most common extra-articular manifestation. The centile charts suggest that females reported greater disease activity and more functional impairment than males but had lower BASMI and mSASSS scores. Data collected through this study provided a demographic and clinical profile of patients with AS in Portugal. The development of centile charts constitutes a useful tool to assess the change of disease pattern over time and in response to therapeutic interventions.

ANKH and susceptibility to and severity of ankylosing spondylitis

Objective. Unconfirmed reports describe association of ankylosing spondylitis (AS) with several candidate genes including ANKH. Cellular export of inorganic pyrophosphate is regulated by the ANK protein, and mutant mice (ank/ank), which have a premature stop codon in the 3′ end of the ank gene, develop severe ankylosis. We tested the association between single-nucleotide polymorphisms (SNP) in these genes and susceptibility to AS in a population of patients with AS. We investigated the role of these genes in terms of functional (BASFI) and metrological (BASMI) measures, and the association with radiological severity (mSASSS). Methods. Our study was conducted on 355 patients with AS and 95 ethnically matched healthy controls. AS was defined according to the modified New York criteria. Four SNP in ANKH (rs27356, rs26307, rs25957, and rs28006) were genotyped. Association analysis was performed using Cochrane-Armitage and linear regression tests for dichotomous and quantitative variables. Analyses of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), BASFI, and mSASSS were controlled for sex and disease duration. Results. None of the 4 markers showed significant single-locus disease associations (p > 0.05), suggesting that ANKH was not a major determinant of AS susceptibility in our population. No association was observed between these SNP and age at symptom onset, BASDAI, BASFI, BASMI, or mSASSS. Conclusion. These results confirm data in white Europeans that ANKH is probably not a major determinant of susceptibility to AS. ANKH polymorphisms do not markedly influence AS disease severity, as measured by BASMI and mSASSS. The Journal of Rheumatology

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10-12) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P tdthomlt; 10-14). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Importance of amino-terminus in maintenance of oligomeric structure of cytosolic serine hydroxymethyltransferase

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1- W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1–V30)-SHMT and des-(A1–L49)-SHMT were expressed at very low levels, whereas des-(A1–R58)-SHMT, des-(A1–G75)-SHMT, des-(Q435–F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1–K6)-SHMT and des-(A1–W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 °C, the des-(A1–W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1–K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1–W14)-SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des-(A1–K6)-SHMT (55 °C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des-(A1–W14)-SHMT compared to rSHMT (1.8 ±0.1 M) and des-(A1–K6)-SHMT (1.7 ±0.1 M). The apoenzyme of des-(A1- W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1–K6)-SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des-(A1–K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1–W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The Role Of The Amino And Carboxyl-Terminal Regions Of Cytosolic Serine Hydroxymethyltransferase (SHMT) In Subunit Assembly And Catalysis Was Studied Using Sis Amino-Terminal (Lacking The First 6, 14, 30, 49, 58, And 75 Residues) And Two Carboxyl-Terminal (Lacking The Last 49 And 185 Residues) Deletion Mutants. These Mutants Were Constructed From A Full Length Cdna Clone Using Restriction Enzyme/PCR-Based Methods And Overexpressed In Escherichia Coli. The Overexpressed Proteins, Des-(A1-K6) SHMT And Des-(A1-W14)-SHMT Were Present In The Soluble Fraction And They Were Purified To Homogeneity. The Deletion Clones, For Des-(A1-V30)-SHMT And Des-(A1-L49)-SHMT Were Expressed At Very Low Levels, Whereas Des-(A1-R58)-SHMT, Des-/A1-G75)-SHMT, Des-(Q435-F483)-SHMT And Des-(L299-F483)-SHMT Mutant Proteins Were Not Soluble And Formed Inclusion Bodies. Des-(A1-K6)-SHMT And Des-(A1-W14)-SHMT Catalyzed Both The Tetrahydrofolate-Dependent And Tetrahydrofolate-Independent Reactions, Generating Characteristic Spectral Intermediates With Glycine And Tetrahydrofolate. The Two Mutants Had Similar Kinetic Parameters To That Of The Recombinant SHMT (Rshmt). However, At 55 Degrees C, The Des-(A1-W14)-SHMT Lost Almost All The Activity Within 5 Min, While At The Same Temperature Rshmt And Des-(A1-K6)-SHMT Retained 85% And 70% Activity, Respectively. Thermal Denaturation Studies Showed That Des-(A1-W14)-SHMT Had A Lower Apparent Melting Temperature (52 Degrees C) Compared To Rshmt (56 Degrees C) And Des-(A1-K6)-SHMT (55 Degrees C), Suggesting That N-Terminal Deletion Had Resulted In A Decrease In The Thermal Stability Of The Enzyme. Further Urea Induced Inactivation Of The Enzymes Revealed That 50% Inactivation Occurred At A Lower Urea Concentration (1.2+/-0.1 M) In The Case Of Des-(A1-W14)-SHMT Compared To Rshmt (1.8+/-0.1 M) And Des-(A1 -K6)-SHMT (1.7+/-0.1 M). The Apoenzyme Of Des-/A1-K6)-SHMT Was Present Predominantly In The Dimer Form, Whereas The Apoenzymes Of Rshmt And Des-(A1-K6)-SHMT Were A Mixture Of Tetramers (Approximate To 75% And Approximate To 65%, Respectively) And Dimers. While, Rshmt And Des-(A1-K6)-SHMT Apoenzymes Could Be Reconstituted Upon The Addition Of Pyridoxal-5'-Phosphate To 96% And 94% Enzyme Activity, Respectively Des-(A1-W14)-SHMT Apoenzyme Could Be Reconstituted Only Upto 22%. The Percentage Activity Refined Correlated With The Appearance Of Visible CD At 425 Nm And With The Amount Of Enzyme Present In The Tetrameric Form Upon Reconstitution As Monitored By Gel Filtration. These Results Demonstrate That, In Addition To The Cofactor, The N-Terminal Arm Plays An Important Role In Stabilizing The Tetrameric Structure Of SHMT.