Recombinant phosphoglucomutase and CAMP factor as potential subunit vaccine antigens induced high protection against Streptococcus iniae infection in flounder (Paralichthys olivaceus).

AIMS: The aim of this study was to screen vaccine candidates from virulence factors of Streptococcus iniae in flounder model. METHODS AND RESULTS: The immunogenicity of recombinant phosphoglucomutase (rPGM) and rCAMP factor was confirmed by Western blot. The percentage of surface membrane immunoglobulin-positive (sIg+ ) lymphocytes in peripheral blood leucocytes, the specific and total serum IgM and the activity of acid phosphatase (ACP) and peroxidase (POD) in flounder were determined with flow cytometry, ELISA and commercial enzyme activity kits, respectively, after intraperitoneal immunization with rPGM and rCAMP factor. The results showed that rPGM and rCAMP factor could induce significant rise in sIg+ lymphocytes, specific serum IgM and activities of ACP and POD. Additionally, the relative percent survival rate of the vaccinated flounder was 64 and 54% in challenge experiment using S. iniae, respectively. These results indicated that rPGM and rCAMP factor could evoke humoural and innate immune response in flounder and provide high-efficiency immunoprotection against S. iniae infection. CONCLUSIONS: Phosphoglucomutase (PGM) and CAMP factor were promising vaccine candidates against S. iniae in flounder. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphoglucomutase and CAMP factor have the potential to be vaccine candidates, which provide important information for us to develop the effective subunit vaccines, especially the multivaccine, against S .iniae in aquaculture.

Primary Immunodeficiencies with Elevated IgE.

In recent years a number of primary immunodeficiencies (PIDs) characterized by elevated Immunoglobulin E (IgE) levels have been uncovered and termed as Hyper-IgE syndrome (HIES). In addition to the elevated levels of IgE, patients with these PIDs display a spectrum of infections by staphylococci and fungi, and in some cases viruses, particularly affecting skin and lungs. Most of these PIDs also have a non-infectious phenotype, comprising musculoskeletal, vascular, and neurological abnormalities. The genetic basis for the majority of conditions with elevated IgE has now been established and includes mutations in STAT3, DOCK8, TYK2, and most recently PGM3 molecules. However, in some patients with the relevant phenotype, mutations in these molecules are not identified, suggesting additional genetic etiologies of HIES not yet discovered. As the immunological and molecular basis of HIES is being unraveled, important insights are emerging that may have implications for our understanding of basic principles of immunology and protective immunity as well as for the pathogenesis and clinical management of patients with these complex and challenging PIDs. In this review, are presented the current knowledge on the clinical presentation, infectious phenotype, and the genetic and immunological pathogenesis of hyper-IgE syndromes as well as some other PIDs with elevated levels of IgE.

Effects of phosphoglucomutase gene (PGM) in Streptococcus parauberis on innate immune response and pathogenicity of olive flounder (Paralichthys olivaceus).

In recent years, Streptococcus parauberis infection has been an emerging problem in aquaculture in South Korea because of its more frequent isolation than other streptococcal bacteria including Streptococcus iniae. To develop effective treatment and prophylaxis methods against this emerging disease by S. parauberis, it is necessary to understand the underlying pathogenic mechanisms. To uncover the pathogenicity, the mutant strain of S. parauberis with a deleted phosphoglucomutase (PGM) gene which has been known to be an important virulence factor in bacterial pathogens was generated to investigate the relationship between virulence and gene function using an allelic exchange mutagenesis method. Allelic exchange mutagenesis of the phosphoglucomutase gene resulted in phenotype changes including decreased extracellular capsules, reduced buoyancy, increased hydrophobicity and reduced growth. Moreover, the S. parauberis mutant was more sensitive to innate immune clearance mechanisms including serum, mucus and phagocyte killing and could not induce mortality in olive flounder. These phenotype changes and the attenuated virulence of the pathogen to fish could be due to the reduction in capsule production by mutation of the PGM gene. The results provide evidences that phosphoglucomutase expression contributes to S. parauberis virulence in fish by affecting bacterial survival against the host's humoral and cellular defense mechanisms.

Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels.

BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.

Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment.

BACKGROUND: Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. OBJECTIVE: We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. METHODS: Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. RESULTS: Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. CONCLUSIONS: Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.

Identification of New Potential Allergens From Nile Perch (Lates niloticus) and Cod (Gadus morhua)

Antecedentes: La globalización de la industria alimentaria proporciona la exposición a nuevos pescados no domésticos y se hace necesaria la identificación de los alérgenos potenciales para diagnosticar las reacciones alérgicas. Objetivo: El objetivo de este estudio fue estudiar las proteínas fijadoras de IgE que constituyen los alérgenos de la perca del Nilo (L. niloticus). Métodos: Mediante electroforesis 2D en gel se separaron las proteínas del músculo del L. niloticus y G. morhua y se enfrentaron al suero de 12 pacientes con historia de reacción inmediata a pescado, así como al suero de pacientes atópicos y controles sanos. Las proteínas reactivas a IgE fueron identificadas mediante espectrofotometría de masas. Results: En los resultados, el paciente mostraba un índice bajo de fijación de IgE a parvalbúminas, sin embargo mostraba fijación de IgE a 8 alérgenos diferentes a la parvalbúmina de L. niloticus y 5 a la G. morhua. Observamos una sensibilización cruzada de 7/12 (58%) de los individuos alérgicos a pescado a la enolasa-3 del L. niloticus, mientras que 11/12 (92%) de los pacientes estaban sensibilizados a la enolasa-3 del G. morhua. Conclusión: La identificación de los alérgenos especie-específicos o de la sensibilización individual podría en el futuro mejorar las estrategias de evitación (AU)

Background: Globalization of the food industry has led to widespread exposure to new nondomestic fish species; therefore, identification of potential allergens is necessary in order to diagnose allergic reactions. Objective: Contact with a patient who was allergic to Nile perch (Lates niloticus) prompted us to investigate the immunoglobulin (Ig) E– reactive proteins that could be allergens of this species. Methods: 2D gel electrophoresis was used to separate the muscle proteins of L niloticus and Gadus morhua. Immunoblotting was performed with sera from 12 patients with a history of immediate-type allergic reaction to fish and from atopic and nonatopic controls. IgE-reactive proteins were detected and identifi ed using mass spectrometry. Results: The index patient had low levels of IgE binding to parvalbumins. However, 8 putative allergens other than parvalbumin from L niloticus and 5 from G morhua were identified. Further investigation revealed cross-sensitivity to enolase 3 from L niloticus in 7 of the 12 fish-allergic individuals (58%), whereas 11 of the 12 patients (92%) were sensitized to enolase 3 from G morhua. However, atopic control patients were also sensitized to enolase 3 from L niloticus and G morhua. Conclusion: Identification of species-specific allergens and individual sensitization could help us to improve avoidance strategies (AU)

Contribution of phosphoglucosamine mutase to the resistance of Streptococcus gordonii DL1 to polymorphonuclear leukocyte killing.

Phosphoglucosamine mutase (GlmM; EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of the peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. We have recently identified the gene (glmM) encoding the enzyme of Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and indicated that the glmM mutation in S. gordonii appears to influence bacterial cell growth, morphology, and sensitivity to penicillins. In the present study, we assessed whether the glmM mutation also affects escape from polymorphonuclear leukocyte (PMN)-dependent killing. Although no differences in attachment to human PMNs were observed between the glmM mutant and the wild-type S. gordonii, the glmM mutation resulted in increased sensitivity to PMN-dependent killing. Compared with the wild type, the glmM mutant induced increased superoxide anion production and lysozyme release by PMNs. Moreover, the glmM mutant is more sensitive to lysozyme, indicating that the GlmM may be required for synthesis of firm peptidoglycans for resistance to bacterial cell lysis. These findings suggest that the GlmM contributes to the resistance of S. gordonii to PMN-dependent killing. Enzymes such as GlmM could be novel drug targets for this organism.

Glucose-6-phosphate isomerase is not a specific autoantigen in rheumatoid arthritis.

OBJECTIVE: To test the hypothesis that glucose-6-phosphate isomerase (GPI) is a novel autoantigen in RA. METHODS: Eighty-eight serum samples from 23 patients with rheumatoid arthritis (RA), 25 with Sjögren's syndrome, 20 with systemic lupus erythematosus and 20 healthy controls were tested by enzyme-linked immunosorbent assay (ELISA) using a commercially available, partially purified rabbit GPI as antigen. Beside each duplicate well containing antigen (10 micro g/ml), uncoated blocked duplicate wells (phosphate-buffered saline only) were included as controls for non-specific binding for every serum tested. We also examined antibodies binding to various polypeptides in the GPI preparation by immunoblotting in 73 of the sera. RESULTS: By ELISA, binding levels were low and there was no difference between serum from patients with RA, other rheumatic diseases and normal controls. By immunoblotting, antibodies binding to the GPI polypeptide were present in 70-80% of all groups tested. In addition, we showed that another polypeptide identified as phosphoglucomutase was also present in the preparation and reacted with human immunoglobulins. CONCLUSION: Our findings suggest that GPI is not a specific autoantigen in RA.

Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity.

PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.

A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase.

We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 ('parafusin') would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.

Antigenic analysis of the major human phosphoglucomutase isozymes: PGM1, PGM2, PGM3 and PGM4.

The cross-reactivity of human phosphoglucomutase isozymes (PGM1, PGM2, PGM3 and PGM4) has been investigated using anti-rabbit muscle PGM polyclonal antibodies. Significant differences were revealed: an IgG fraction of the antiserum reacted with the primary and secondary PGM1 isozymes of all the common phenotypes. However, there was no reaction with the PGM2 or PGM3 isozymes; thus these latter isozymes share no major antigenic determinants with human or rabbit PGM1 and are therefore structurally distinct. In contrast, the PGM isozymes of human milk attributed to a fourth locus, PGM4, showed similar cross-reactivity as PGM1 suggesting close structural similarity. The IgG was also employed as a reagent to remove PGM1 from haemolysates so as to allow the unambiguous assessment of the PGM2 isozyme patterns by isoelectric focusing. However, no proven genetic variation was encountered in a sample of 32 individuals.

Immunological studies on erythrocyte phosphoglucomutase isozymes.

Human erythrocytes (phenotype PGM1 a1 or PGM1 a3) contain two sets of phosphoglucomutase isozymes, produced by the expression of the PGM1 and and PGM2 loci. The two sets are constituted each by two forms, of which that called "secondary" is thought to derive from the post-translational modification of that called "primary". Cross-reactivities of these isozymes were studied by means of monospecific rabbit antibodies against purified human red cell PGM1 and PGM2 "primary" isozymes. The results show that the PGM1 and PGM2 forms are not immunologically related and provide further proof of the post-synthetic origin of "secondary" isozymes and of the multifunctionality of PGM2 phosphoglucomutases.

Demonstration of antibodies to phosphoglucomutase of parasitic origin in Brugia pahangi-infected cats.

Antibodies inhibitory to the activity of the enzyme phosphoglucomutase (PGM) EC 2.7.5.1. of Brugia pahangi adult worm were demonstrated in sera from five rabbits immunized against this filarioid and from 5 of 27 cats infected with B. pahangi, by isoelectricfocusing and spectrophotometric techniques. This anti-PGM activity was species- and stage-specific. This raises the possibility of using species-specific isoenzymes of parasitic origin as antigens in serological tests.

Association of blood groups with essential and secondary hypertension. A possible association of the MNS system.

Persons participating in a 5-day diagnostic protocol were routinely typed for ABO, Rh, MNS, Kell, Kidd, Duffy, P, Haptoglobin, phosphoglucomutase-1 (PGM-1), and acid phosphatase (AcP). The study population was composed of 164 normotensive whites, 34 normotensive blacks, 161 whites and 43 blacks with essential hypertension, and 52 whites with secondary forms of hypertension (18 atherosclerotic renovascular hypertensives, 17 patients with fibromuscular disease, and 17 patients with primary aldosteronism). There were no significant differences in phenotype frequencies in ABO, Rh, Kidd, Kell, Duffy, P, Haptoglobin, PGM-1 or AcP in any of the comparisons. However, there was a significantly different distribution of MNS phenotypes in comparisons of essential and atherosclerotic renovascular hypertensives with normotensive controls. Essential hypertensives had a lower frequency of the S gene and a higher frequency of s in whites (X2 = 12.21, p less than 0.005). Atherosclerotic renovascular hypertensives differed from the normotensive population in the frequencies of both MN (X 2 = 4.34, p less than 0.05) and Ss (X2 = 4.21, p less than 0.05). The finding of disease-blood group associations supports the hypothesis that there may be significant physiological differences between individuals of different blood types.

Sero-genetic studies on the Dama of South West Africa.

The Dama of South West Africa are a Negroid people living as a reproductive isolate in the desert and semi-desert areas of the north-west of the country. Until recent times a large proportion of them were held in bondage by the Khoikhoi (Hottentot) Nama, while the rest lived as hunter-gatherers in the mountains. This study and the work of Knussmann and Knussmann indicate that they are a Negro people, which probably has been cut off over a period from contact with other Negroes. They have received very little genetic contribution from the Khoikhoi or the San (Bushmen). The results of this investigation of 24 blood genetic marker systems in a carefully selected random sample of Dama support these conclusions.