Developments in the field of allergy in 2014 through the eyes of Clinical and Experimental Allergy.

The pathogenesis of asthma continues to be a major topic of interest to our authors with reviews and original papers on the role of viruses, mechanisms of inflammation, biomarkers, and phenotypes of asthma being major topics. A number of papers described new treatments for asthma focusing on blocking the Th2 response reflecting the fact that two decades of work in this area is finally bearing fruit. The pathogenesis of chronic rhinosinusitis is a growing area of interest, but there has been less on the genetics of airways disease than in previous years possibly reflecting the degree of rigour (and therefore a smaller body of work), with which these sorts of studies are now being undertaken. There continues to be a wide range of papers dealing with mechanisms of allergic disease ranging from clinical-based studies to basic research and the use of in vivo animal models especially mice. As before, mechanisms and new approaches to immunotherapy are common themes. Several were published in the allergens section investigating modification of allergens to increase their effectiveness and reduce the risk of adverse events. Risk factors for allergic disease was a common theme in the epidemiology section and food allergy a common theme in clinical allergy with papers on the development of protocols to induce tolerance and attempts to find biomarkers to distinguish sensitization from allergic disease. This was another exciting year for the editors, and we hope the readers of the journal.

Genome-wide association study of IgG1 responses to the choline-binding protein PspC of Streptococcus pneumoniae.

Streptococcus pneumoniae causes invasive pneumococcal disease. Delayed development of antibodies to S. pneumoniae in infancy is associated with the development of atopy and asthma. Pneumococcal surface protein C (PspC) is a vaccine candidate and variation in its choline-binding region is associated with invasive disease. This study examined 523 060 single-nucleotide polymorphisms in The Western Australian Pregnancy Cohort (Raine) Study to find loci influencing immunoglobulin G1 (IgG1) responses to PspC measured at age 14 years (n=1152). Genome-wide significance (top SNP rs9275596; P=3.1 × 10(-14)) was only observed at human leucocyte antigen (HLA). Imputed HLA amino-acid polymorphisms showed the strongest associations at positions DRB1 47 (P=3.2 × 10(-11)), 13SRG (P=9.8 × 10(-10)) and 11SP (P=9.8 × 10(-10)), and at DQA1 34 (P=6.4 × 10(-10)), DQB1 167R (P=9.3 × 10(-6)) and HLA-B 95 W (P=1.2 × 10(-9)). Conditional analyses showed independent contributions from DRB1 47 and DQB1 167R to the signal at rs9275596, supported by an omnibus test showing a strong signal for the haplotype DRB1_47_DQB1_167 (P=9.02 × 10(-15)). In silico analysis showed that DRB1 four-digit allele groups defined by DRB1 47F bind to a greater complexity of core 9-mer epitopes compared with DRB1 47Y, especially across repeats in the C-term choline-binding region. Consequent differences in CD4 T-cell help for IgG1 to PspC could have implications for vaccine design. Further analysis in other cohorts is merited.

Lower anti-echovirus antibody responses in children presenting to hospital with asthma exacerbations.

BACKGROUND: Rhinoviruses from the Enterovirus genus cause frequent infections and induce remarkably high titres of anticapsid antigen antibodies in asthmatics, while the prevalence of neutralising antibodies to the gut-trophic echoviruses from the same genus is diminished. OBJECTIVE: To assess the absolute and specific antibody titres to VP1 antigens of the gut-trophic enteroviruses, echovirus 30 and Sabin 1 poliovirus, in asthmatic and non-asthmatic children. METHODS: Recombinant polypeptides representing the VP1 capsid antigens of echovirus 30 and Sabin poliovirus 1 were produced. Their ability to bind IgG1 antibodies from the plasma of asthmatic (n = 45) and non-asthmatic (n = 29) children were quantitated by immunoassays that incorporated immunoabsorptions to remove cross-reactivity. RESULTS: The IgG1 antibody titres and prevalence of antibody binding to echovirus 30 were significantly lower for asthmatic children compared to controls (P < 0.05) and inversely correlated with total IgE levels for the whole study population (r = -0.262; P < 0.05). There was no difference in the prevalence and titre between groups to the VP1 antigen of Sabin poliovirus. Anti-tetanus toxoid titres measured for comparison did not correlate with anti-echovirus or poliovirus, but correlated with anti-rhinovirus titres in controls but not asthmatics, where the titres were higher for the asthmatic group. CONCLUSIONS AND CLINICAL RELEVANCE: The associations of lower antibody titres of asthmatic children to echovirus reported here and those of our previous findings of a heightened response to rhinovirus suggest a dichotomy where respiratory enterovirus infection/immunity increases the probability of developing asthma and enteric infections lower the risk. This provides further support for the concept of intestinal infection playing a key role in the development of allergic respiratory disease.

A role for CCL28-CCR3 in T-cell homing to the human upper airway mucosa.

Lymphocyte recruitment to peripheral tissues is fundamental for immune surveillance and homeostasis, but the chemokines and chemokine receptors responsible for tissue-specific homing of T cells to the upper airway mucosa have not been determined. To address this, we analyzed the chemokines expressed in the normal human nasal mucosa and found that CCL28 is preferentially expressed at a high level on the lumenal face of vascular endothelial cells in the mucosa. Analysis of the cognate chemokine receptors revealed that close to 50% of the CD4(+) T cells in the human nasal mucosa expressed the CCL28 receptor CCR3, whereas CCR3 was hardly detectable on T cells in the small intestine and skin. In the circulation, CCR3(+) T cells comprised a small subset that did not express homing receptors to the intestine or skin. Moreover, depletion of CCR3(+)CD4(+) T cells abrogated the proliferative response of human blood CD4(+) T cells against the opportunistic nasopharyngeal pathogen Haemophilus influenzae, indicating that the CCR3(+)CD4(+) T-cell subset in the circulation contains antigen specificities relevant for the upper airways. Together, these findings indicate that CCL28-CCR3 interactions are involved in the homeostatic trafficking of CD4(+) T cells to the upper airways.

Two newly identified cat allergens: the von Ebner gland protein Fel d 7 and the latherin-like protein Fel d 8.

INTRODUCTION: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. MATERIALS AND METHODS: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. RESULTS: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. DISCUSSION: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

Th2-associated immunity to bacteria in teenagers and susceptibility to asthma.

Bacterial colonisation of the airways is associated with increased risk of childhood asthma. Immunoglobulin (Ig)E against bacterial antigens has been reported in some asthmatics, suggesting a role for bacterial-specific type-2 immunity in disease pathogenesis. We aimed to investigate relationships between bacterial-specific IgE amongst teenagers and asthma susceptibility. We measured titres of IgE against Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus in 1,380 teenagers, and related these to asthma symptomatology and immunophenotypes. IgE titres against S. aureus-derived enterotoxins were highest amongst atopics and were associated with asthma risk. Surprisingly, IgE titres against H. influenzae and S. pneumoniae surface antigens were higher, not stratified by atopy and independently associated with decreased asthma risk. The positive association between type-2 immunity to S. aureus and asthma phenotypes probably reflects IgE-mediated effector cell activation via enterotoxin super antigens which are secreted in soluble form. The contrasting benign nature of type-2 immunity to H. influenzae and S. pneumoniae antigens may reflect their lower availability in soluble forms that can crosslink IgE receptors. We theorise that instead they may be processed by antigen presenting cells and presented to type-2 memory cells leading to mucosal secretion of interleukin (IL)-4/IL-13, a mechanism widely recognised in other tissues to attenuate T-helper-1 associated bacterial-induced inflammation.

Anti-bacterial IgE in the antibody responses of house dust mite allergic children convalescent from asthma exacerbation.

BACKGROUND: Atopic sensitization to the house dust mite (HDM) is associated with altered antibody responses to the nasopharyngeal colonizing bacterium Haemophilus influenzae and children admitted to the emergency department for asthma exacerbation have reduced IgG responses to HDM allergens. OBJECTIVE: To investigate anti-bacterial and anti-allergen antibody responses during convalescence from asthma exacerbation and differences found in exacerbations associated with and without viral infection. RESULTS: IgE antibodies to the P6 bacterial antigen increased in 60% of sera during convalescence and for many children achieved titres as high as IgE titres to allergens. In contrast IgE anti-HDM titres declined during convalescence. The anti-bacterial IgE titres were the same in subjects with and without virus infection while the anti-HDM IgE declined more rapidly in virus-infected subjects. IgG titres to the major HDM allergens showed no consistent increase and the overall IgG anti-HDM titres even declined in subjects without a virus infection. Anti-bacterial IgG antibodies in contrast to IgE did not change. Patients with frequent episodic or persistent asthma had similar IgE anti-bacterial titres to patients with infrequent asthma during the acute phase, although they had reduced IgG titres to both the bacteria and the HDM. CONCLUSIONS: During the period following an acute exacerbation of asthma there was a marked and specific increase in anti-bacterial IgE compared with a reduced IgE response to HDM. This provides further support for the concept of T-helper type 2 responses to bacterial antigens playing a role in asthma pathogenesis.

Differences in the antibody response to a mucosal bacterial antigen between allergic and non-allergic subjects.

BACKGROUND: The immune response to bacterial antigens on mucosal surfaces may be modified in individuals allergic to aeroallergens due to a maturational or genetic difference or from the interaction between inhaled allergens and bacteria at the mucosa. METHODS: Plasma from children and adults allergic (n = 97) and non-allergic (n = 54) to aeroallergens were initially tested for IgG1 (Th1) and IgG4 (Th2) reactivity to P6, a conserved outer membrane protein of Haemophilus influenzae. IgE binding was measured for some allergic donors. The development of the antibody responses to P6 was subsequently examined in the plasma from 35 children aged 1, 2 and 5 years taken from a prospective birth cohort. RESULTS: IgG4 antibodies to P6 were more readily detected in allergic subjects than in non-allergic subjects (p<0.001), with a strong bias to the male gender. Some allergic subjects (35%) also had IgE antibody (1-10 ng/ml) that was not associated with IgG4 or gender. In the cohort study of infants, subjects who developed skin prick test positivity to mite allergens by 5 years of age had an 85% reduction in the IgG1 anti-P6 antibody at year 2 (p<0.05) and, unlike skin test negative infants, this group had IgG4 anti-P6 antibodies at 5 years of age. CONCLUSIONS: The antibodies of subjects allergic to a bacterial antigen included IgE and IgG4 (particularly for males) compared with the almost exclusive IgG1 response of non-allergic subjects. The IgG1 responses of 2-year-old children who became skin test positive was markedly reduced and P6-specific IgG4 became detectable at 5 years of age.

Distinctive immunoglobulin E anti-house dust allergen-binding specificities in a tropical Australian Aboriginal community.

BACKGROUND: There is evidence that the specificity of the IgE binding in allergy tests can vary for different populations. OBJECTIVE: We aimed to examine the allergenic specificity of IgE binding in sera from house dust mite (HDM)-atopic subjects in a tropical Australian Aboriginal community. METHODS: Sera shown to contain IgE antibodies to an HDM extract of Dermatophagoides pteronyssinus were examined for IgE binding to a panel of nine purified HDM allergens from this mite species by quantitative microtitre assays. IgG antibody binding (IgG1 and IgG4) was also measured. RESULTS: The IgE-binding activity in the sera from the Aboriginal community was not directed to the expected major groups 1 and 2 HDM allergens but instead to the group 4 amylase allergen. There was also little IgE binding to the potentially cross-reactive tropomyosin (Der p 10) or arginine kinase (Der p 20) allergens. The IgG4 antibody was rarely detected and limited to the Der p 4 allergen. IgG1 antibody binding was frequently measured to all the allergens regardless of an individual's atopic status, whereas in urban communities it is restricted to the major allergens and to atopic subjects. CONCLUSION: The high IgE anti-HDM response of Australian Aboriginals predominantly bound Der p 4 and not the Der p 1 and 2 allergens, showing a distinctive allergy that could affect the disease outcome and diagnosis.

The chitinase allergens Der p 15 and Der p 18 from Dermatophagoides pteronyssinus.

BACKGROUND: House dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae cause allergic disease in humans as well as in dogs. In geographical regions where the two mite species coexist, they both elicit specific immunoglobulin (Ig E) responses in humans whereas dogs preferentially react to D. farinae extracts. In dogs the main IgE binding is directed to the D. farinae chitinase allergens Der f 15 and Der f 18 and not to the groups 1 and 2 allergens as found for humans. Although the IgE response of humans to Der f 18 has been investigated there is no report on Der f 15-specific IgE in humans. OBJECTIVE: This study aimed to characterize the chitinase allergens Der p 15 and Der p 18 of D. pteronyssinus and to find out whether they are important allergens for humans. METHODS: cDNA was cloned by a polymerase chain reaction strategy from D. pteronyssinus libraries using primers based on conserved chitinase sequences. IgE binding to the recombinant polypeptides was measured by immunosorbent assay. Mice were immunized with the polypeptides and cross-reactivity examined. RESULTS: Two variants of Der p 15 were isolated, encoding mature proteins of 58.8 and 61.4 kDa. The amino acid sequences had 90% identity to Der f 15. The cDNA for Der p 18 encoded a mature protein of 49.2 kDa with 88% sequence identity to Der f 18. Der p 15-specific IgE was detected in 70% and Der p 18-specific IgE in 63% of a panel of 27 human allergic sera. CONCLUSIONS: The D. pteronyssinus chitinases Der p 15 and Der p 18 show a high frequency of binding to IgE in allergic human sera. They are therefore potentially important allergens for humans as well as dogs.

Identification of tropomyosin, paramyosin and apolipophorin/vitellogenin as three major allergens of the sheep scab mite, Psoroptes ovis.

Analysis by one-dimensional (1-D) SDS-PAGE/Western blotting of whole mite extract (larval and adult stages) with sheep sera taken 0-84 days after infection with the sheep scab mite, Psoroptes ovis revealed a progressive IgE antibody response, with a dominant high molecular weight allergen (MW 180 kDa) detected early during infection. Further analysis by two-dimensional (2-D) SDS-PAGE/Western blotting with post-infection sera, revealed a more complex picture with numerous (> 20) IgE reactive spots. Trypsin digest and Maldi-ToF analyses of these spots identified two house dust mite allergen homologues, namely tropomyosin (Der p 10) and paramyosin (Der p 11), and analysis of a third spot indicated an apolipoprotein-like IgE reactive protein (Der p 14). Further 1-D and 2-D SDS/Western blotting, with specific antibodies to the house dust mite allergens Der p 10, 11, and to the IgE reactive peptide of the high molecular weight house dust mite allergen, Der p 14 (vitellogenin/apolipophorin), provided firm evidence for the presence of these three allergens in extracts of the Psoroptes mite. These studies show for the first time that homologues of the house dust mite 10, 11 and 14 group allergens are represented as immunodominant allergens of the sheep scab mite, and may represent important vaccine candidates.

Genetic variation of Der p 2 allergens: effects on T cell responses and immunoglobulin E binding.

BACKGROUND: Der p 2 is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variations on T cell and antibody responses has not been compared. OBJECTIVES: To compare IgE antibody binding and T cell proliferation and cytokine release induced by variants of Der p 2. METHODS: Peripheral blood mononuclear cells (PBMC) from 19 allergic and 15 non-allergic people were stimulated with recombinant variants of Der p 2. IL-5, IL-10, IL-13 and IFN-gamma were measured by a time resolved fluorescence (TRF) assay. Serum IgE antibody was measured using a solid-phase TRF assay. RESULTS: Overall the most prevalent variant of Der p 2 (Der p 2. 0101) was the highest or approximately equal highest inducer of T cell proliferation and IL-5, IL-10, IL-13 and IFN-gamma release. The most divergent variant 0104 induced the next highest responses. The variants 0107 and 0108 showed interesting changes especially when the allergic status was considered. Responses to 0107 showed poor Th1/Th2 polarization and, except for IL-10 release, cytokine responses to 0108 were low for non-allergic subjects. The variant 0101 showed similar monoclonal antibody binding but moderately less IgE binding than the other variants. CONCLUSIONS: The most prevalent variant, Der p 2. 0101, was the most active for T cell stimulation and although its IgE binding was slightly less than other variants that was highly correlated. The variant Der p 2. 0104 which contains the known common polymorphic changes had a response which was similar to Der p 2. 0101 and thus these two variants were the most stimulatory representations of Der p 2. The T cell responses to the less common variants 0107 and 0108 however, showed consistent differences demonstrating that changes in the sequence could change the cytokine response.

Non-allergenic antigen in allergic sensitization: responses to the mite ferritin heavy chain antigen by allergic and non-allergic subjects.

BACKGROUND: The majority of house dust mite proteins are non-allergenic. There is, however, no information on the type of immune responses produced to these proteins and if the responses are affected by allergic sensitization. OBJECTIVE: To identify and produce a non-allergenic antigen of the house dust mite and compare antibody and T cell responses with the responses to allergens in sensitized and non-sensitized individuals. RESULTS: Ferritin heavy chain was cloned from a cDNA library as a candidate non-allergen of the house dust mite. It bound IgG but not IgE in the sera of allergic and non-allergic subjects and induced high T cell proliferative responses that correlated highly with the responses to the major allergen Der p 2. The cytokine response to the non-allergen was characterized by the release of high levels of both Th1 and Th2 cytokines from the PBMC of both allergic and non-allergic subjects. In contrast, the response to Der p 2 showed the expected high level of Th2 cytokine release from the PBMC of allergic subjects, while the Th2 cytokine production from PBMC of non-allergic subjects was low and even lower than that induced by ferritin heavy chain. The levels of IFN-gamma release were similar for all groups. Der p 2 induced significantly more IL-10 than ferritin in the non-allergic group. CONCLUSION: The T cell responses to a non-allergenic protein of the house dust mite were high and strongly correlated with the response to the major allergen. The non-allergenic protein induced high levels of Th1 and Th2 cytokine in both allergic and non-allergic subjects, while the allergen induced high levels of Th2 cytokine in allergic subjects and low levels in non-allergic subjects. The responses to the allergen were thus independently up- and down-regulated with no evidence of bystander regulation.

T cell cytokine responses to outer membrane proteins of Haemophilus influenzae and the house dust mite allergens Der p 1 in allergic and non-allergic subjects.

BACKGROUND: Haemophilus influenzae are ubiquitous colonizers of the nasopharynx, Little is known about the T cell cytokine responses to the antigens of these bacteria and whether or not the responses may interact with responses to aeroallergen. OBJECTIVE: To measure the T cell cytokine responses to conserved outer membrane protein antigens of Haemophilus influenzae and to house dust mite allergen of subjects allergic to the house dust mite and of subjects without allergic sensitization. METHODS: T cell responses were measured by in vitro proliferation and cytokine release from peripheral blood monocytes (PBMC). The allergen used was Der p 1 and outer membrane proteins were recombinant polypeptides representing the OMP6 and D15 antigens. RESULTS: The PBMC of most subjects had proliferative responses to OMP6 and D15, which were highly correlated. The pattern of cytokine release was Th1 biased with high levels of IFN-gamma and usually little IL-5 or IL-13 although PBMC from a few subjects did release IL-5 independent of allergic status. IL-10 release was readily detected. There was no difference in the anti-OMP cytokine response of PBMC from subjects without any known allergy and the responses of PBMC from subjects who were highly allergic to house dust mite. The responses to the Der p 1 allergen showed the expected Th2 cytokine release. CONCLUSION: The outer membrane protein antigens of the ubiquitous colonizing bacteria Haemophilus influenzae induce Th1 cytokine responses which are similar for PBMC from non-allergic individuals and subjects with a high degree of allergy to the perennial house dust mite allergen and strong Th2 responses to Der p 1.

Allergens of wild house dust mites: environmental Der p 1 and Der p 2 sequence polymorphisms.

BACKGROUND: Sequence diversity is a common feature of mite allergens. Previous studies, using predominantly commercial mite clones, have described several polymorphic residues for Der p 1 and Der p 2. OBJECTIVE: This study aimed at determining the occurrence of sequence diversity in environmental mite isolates. METHODS: Mites were isolated from houses in Perth and Sydney, Australia. Total RNA was extracted from 1 to 30 Perth mites, and cDNA was synthesized by reverse transcriptase PCR. Der p 1 and Der p 2 cDNAs were PCR amplified and sequenced. Genomic Der p 1 DNA was amplified from whole Sydney mites directly by PCR and then sequenced. RESULTS: Twelve Der p 1 and 9 Der p 2 cDNA clones and 3 Der p 1 genomic DNA were analyzed and showed a high frequency of amino acid polymorphisms. Der p 2 displayed a clear pattern of divergence toward 2 alleles that differed by 4 amino acids and had characteristic silent nucleotide changes. The pattern for Der p 1 was different and unusual, with almost no silent nucleotide substitutions but frequent sporadic missense changes. Proliferative responses of peripheral blood mononuclear cells to peptides containing polymorphic residues of Der p 1 were detected in 8 of 19 subjects, with stimulation being found only for either one of the variant forms of the peptides. However, the responses to variants of whole recombinant allergens were similar, as shown for 4 variants of Der p 2. CONCLUSION: Two clones for each of the allergens were identified as containing sequences that were largely representative of environmental isolates. A small-scale reverse transcriptase PCR used to produce cDNA from individual mites isolated from house dust will have wide application for studies on mite genetics and the production of recombinant mite allergens. Differences in T-cell responses to peptides representing variant epitopes were found, but responses to variants of whole recombinant allergens were similar. The GenBank and Swiss Prot database entries for Der p 1 (U11695) and Der p 2 (P49278) have been updated with the inclusion of the sequence polymorphisms described in this study.

Mechanistic features and structure of the nitrogenase alpha-Gln195 MoFe protein.

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [Sørlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.