Molecular profiling of the immune response in colon cancer using protein microarrays: occurrence of autoantibodies to ubiquitin C-terminal hydrolase L3

We implemented a protein microarray approach to identify proteins that induce a humoral response in colon cancer. Solubilized proteins from the LoVo colon adenocarcinoma cell line were separated into 1760 fractions, arrayed onto nitrocellulose-coated slides, and hybridized with individual sera from 15 newly diagnosed patients with colon cancer, 15 with lung cancer, and 15 healthy subjects. 39/1760 fractions showed enhanced reactivity with sera from patients with colon cancer (p < 0.01) relative to healthy controls. A distinct pattern of reactivity was observed with sera from colon cancer relative to lung cancer. One fraction that exhibited reactivity with 9/15 colon cancer sera was subjected to mass spectrometry leading to the identification of ubiquitin C-terminal hydrolase isozyme 3 (UCH-L3) as a constituent. To validate the occurrence of autoantibodies to UCH-L3, independent analysis was done by means of Western blots. UCH-L3 antibodies were detected in 19/43 sera from patients with colon cancer, and in 0/54 sera from subjects with lung cancer (24), colon adenoma (15) or otherwise healthy (15). Our findings indicate the occurrence of an immune response to a broad set of antigens in colon cancer and the feasibility of identifying the antigenic targets using a combination of protein microarrays and mass spectrometry.     

大疱性类天疱疮IgG型抗基底膜带抗体的靶抗原初步分析

目的　研究大疱性类天疱疮 Ｉｇ Ｇ 型抗基底膜带抗体的靶抗原。方法　应用 Ｄ Ｉ Ｆ与 Ｉ Ｉ Ｆ检测的 Ｉｇ Ｇ型抗体阳性 Ｂ Ｐ 血清进行 Ｗ ｅｓｔｅｒｎ 免疫印迹分析。结果　初步研究发现 Ｉｇ Ｇ 阳性的 Ｂ Ｐ 血清能结合抗原１８０ Ｋ Ｄ,１６０ Ｋ Ｄ 以及 １０５ Ｋ Ｄ 与 ７７ Ｋ Ｄ 的多肽。结论　 Ｉｇ Ｇ 型 Ｂ Ｐ自身抗体可能靶向基底膜带不同层出现的不同抗原并提示该病的发生可能涉及不同的免疫机制。     

Proteomics-based identification of human acute leukemia antigens that induce humoral immune response

The identification of panels of tumor antigens that elicit an antibody response may have utility in cancer screening, diagnosis, and establishing prognosis. Until now, autoimmunity in cancer has been mainly revealed in solid tumors. The aim of this study was to apply the proteomic approach to the identification of proteins that commonly elicit a humoral response in acute leukemia (AL). Sera from 21 newly diagnosed patients with AL, 20 patients with solid tumors, and 22 noncancer controls were analyzed for antibody-based reactivity against AL proteins resolved by two-dimensional electrophoresis. As a result, autoantibody against a protein identified by mass spectrometry as Rho GDP dissociation inhibitor 2 was detected in sera from 15 of 21 patients with AL (71%). By contrast, such antibody was detected in sera from one of 20 patients with solid tumors (5%) and one of 22 noncancer controls (4.5%). Five other protein autoantibodies were also found in AL patients with a high frequency and constituted the major target antigens of the AL autoimmune response. The findings of autoantibodies against Rho GDP dissociation inhibitor 2 and other proteins in sera of patients with AL suggest that the proteomic approach we have implemented may have utility for the development of a serum-based assay for AL screening and diagnosis.     

The incidence of the pituitary autoantibodies in Graves' disease

INTRODUCTION: It is known that in the sera of patients with Graves, Addison and other autoimmune endocrine diseases we can detect autoantibodies against pituitary antigens. The aim of the study was evaluation of pituitary autoantibodies in Graves' disease patients using immunoblotting methods. MATERIAL AND METHODS: Studies were performed in 32 Graves' disease patients, 25 women (age range: 31-67 yrs, median: 49.9 +/- 9.4) and 7 men (age range: 41-58 yrs, median: 51.0 +/- 7.1). All patients presented signs and symptoms typical of thyrotoxicosis. The diagnosis was confirmed by laboratory tests (TSH, fT(3), fT(4), TSH-R antibodies). Sera of control subjects were obtained from 10 healthy blood donors, 7 women, 3 men (age range 21-45 yrs, median: 30.6 +/- 7.1). Incidence of pituitary autoantibodies was assessed by polyacrylamide electrophoresis gel and western-blotting. Pituitary microsomes were obtained from human pituitary tissues by ultracentrifugation and solubilisation in 1% desoxycholic acid. RESULTS: In 23 sera from 32 we detected autoantibodies against pituitary microsomal antigens. 16 sera were reacting with 55 kDa antigen, 10 sera with 67 kDa, 6 sera with 60 kDa, 5 sera with 52 kDa and 4 sera with 105 kDa. It is important to note that 6 sera were reacting with 57 and 55 kDa, and 5 sera with 55, 60 and 67 kDa. CONCLUSIONS: In sera of Graves' disease patients autoantibodies against pituitary microsomal antigens can be frequently detected. The most frequent are antibodies against 55, 60 and 67 kDa antigens.     

Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens

The detection of autoantibodies to tumor antigens has potential utility for the early diagnosis of cancers. In previous studies, we have identified tumor antigens based on Western blot analysis of tumor cell lysates that were incubated with subject sera to identify proteins that elicit specific reactivity in sera from patients with the corresponding tumor type. More recently, we have explored the use of microarrays spotted with tumor proteins as an alternative to Western blots. Microarrays provide a high throughput, high sensitivity alternative to the use of Western blots for tumor antigen profiling. In this study, we have assessed the reproducibility of natural protein microarrays and their ability to distinguish between lung cancer sera and controls. Protein lysates from the A549 human lung adenocarcinoma cell line were separated into 1840 fractions that were spotted in duplicate, along with various controls, on nitrocellulose coated slides. Sera from 18 newly diagnosed patients with lung cancer and from 15 healthy controls were each hybridized to an individual microarray. The reactivity of arrayed proteins with Ig was determined by incubation with biotinylated goat-anti-human-Ig followed by phycoerythrin-conjugated streptavidin. The intensity measures of duplicate spots (within-slide) and duplicate slides (between-slides) were highly reproducible, exhibiting correlation values >0.9. A total of 63 of the 1840 arrayed fractions demonstrated increased reactivity in cancer patients relative to controls as measured by a rank-based statistic (p < 0.008). Microarrays of tumor-derived proteins provide the means for uncovering a repertoire of tumor antigens that have induced an antibody response in patients with specific cancers.     

Characterization of human malignant melanoma cell lines. III. Membrane immunofluorescence reactivity with sera from patients with melanoma

Summary Seven well-characterized human malignant melanoma cell lines have been evaluated in terms of their reactivity in membrane immunofluorescence tests with sera from 48 patients with melanoma, 23 patients with other forms of cancer and 28 normal controls. There was a significantly greater degree of reactivity of melanoma sera (33.7%) than of sera of normal controls (22.2%) or of sera from patients with other forms of cancer (24.2%). The incidence of strong reactors among the melanoma patients was found to be inversely proportional to the extent of disease in the melanoma patients: Stage I, 54.5%, Stage III, 36.8% and Stage IV, 29.4%. Reactivity against non-melanoma cell lines was similar in the three subject groups and was unaffected by stage of disease in the melanoma patients. No single cell line showed preferential reactivity with melanoma sera. There was an increased overall incidence of reactivity of all three subject groups against non-pigmented cell lines.A-B-0 antigens and heterophile antigens were excluded as a cause of seropositivity. The antigen(s) was trypsin-sensitive and neuraminidase-resistant.These data suggest that long term cultures of human melanoma may contain melanoma-associated antigens which may be useful in the further study and search for melanoma-specific antigens.     

Identification of target antigens of anti-endothelial cell and anti-vascular smooth muscle cell antibodies in patients with giant cell arteritis: a proteomic approach

Introduction

Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens.

Methods

Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs). Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs. Target antigens were identified by mass spectrometry.

Results

Serum IgG from GCA patients recognised 162 ± 3 (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively. In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs. Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways. Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein 2.

Conclusions

IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis. Their potential pathogenic role remains to be determined.     

Preferential expression of the systemic lupus erythematosus-associated idiotype 8.12 in sera containing monoclonal immunoglobulins

The 8.12 idiotype defines a population of anti-DNA antibodies present in the serum of patients with systemic lupus erythematosus. As part of our studies to elucidate the genetic origin and structural features of anti-DNA antibodies, we examined monoclonal immunoglobulin (Ig)-containing sera from 706 patients for expression of the 8.12 idiotype. We found 41 such sera to have significant 8.12 reactivity (greater than 4 SD above the mean of normal controls) and demonstrated that in 24 of these sera (8 IgM, 14 IgG, and 2 IgA) this reactivity could be localized to the monoclonal protein. In addition, 12 of the 8.12-reactive monoclonal Ig (11 IgG and 1 IgA) bind dsDNA. In the other 17 sera, the 8.12 reactivity could be attributed to polyclonal antibody. These findings provide further evidence that the serum monoclonal Ig frequently express the antigenic and idiotypic reactivities of autoantibodies. Furthermore, these data support the contention that anti-DNA specificity may result from somatic diversification of germ-line Ig gene sequences.     

Anti-human cardiac myosin autoantibodies in Kawasaki syndrome.

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.     

New autoantibodies in early rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease causing articular cartilage and bone destruction. Since irreversible joint destruction can be prevented by intervention at the early stages of disease, early diagnosis of RA is important. In this study, we identified new autoantibodies in the sera of patients with early (less than one year) RA.

Methods

We screened the sera of 20 RA patients with disease duration less than one year, 19 RA patients with disease duration more than five years and 23 controls on 8,268 human protein arrays. We confirmed the validity of protein array detection by ELISA assays. We then performed epitope mapping with overlapping 15-mers to analyze RA sera reactivity.

Results

WIBG (within BGCN homolog (Drosophila)), GABARAPL2 (GABA(A) receptor associated protein like 2) and ZNF706 (zinc finger protein 706) proteins are preferentially recognized by autoantibodies from early RA patients. Of interest, autoantibodies to WIBG are very specific for early RA. Indeed, 33% of early RA patients'' sera recognize WIBG versus 5% of RA patients with disease duration more than 5 years and 2% of controls. We identified three linear peptides on WIBG GABARAPL2 and ZNF706 that are preferentially recognized by sera of early RA patients.

Conclusions

We identified new autoantibodies associated with RA with disease duration less than one year. These autoantibodies could be used as diagnosis markers in RA patients.     

Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients with different clinical forms of paracoccidioidomycosis

Serum antibodies against antigens of Paracoccidioides brasiliensis have been one of the major diagnostic indicators of paracoccidioidomycosis (PCM). In the present study, released antigen preparations (exoAg) obtained from P. brasiliensis isolates were characterized in terms of their protein components electrophoretically detectable and recognizable by sera (IgG) of patients. Among five different isolates (DGO, C-9, BAT, Pb-18 and B-339) the electrophoretic profiles of exoAg varied greatly. A total of 28 different components were detected, 11 of them shared by all isolates. The most representative preparation was BAT-exoAg, which presented the largest number of protein bands (23) and the highest frequency of reacting bands (19) with sera from patients with active PCM (n = 40). Six bands reacted with more than 20% of sera. Independently of clinical forms, the sera recognized the 43-kDa (97% of tested sera), 160-kDa (78% of tested sera) and 70-kDa (60% of tested sera) antigens more frequently. Sera from patients with severe forms of acute (n = 14) or chronic (n = 10) PCM recognized a greater number of antigens, with a higher frequency, than those from moderate forms. The most pronounced reduction in reactivity was provided by sera of patients that became asymptomatic at the beginning of treatment. Remnant reactivity with BAT-exoAg was detected after clinical recovery, especially with those of 43, 70 and 160 kDa. The latter presented a stable recognition frequency (60%) during the entire follow-up, allowing us to suppose that the IgG reactivity against the 160-kDa antigen constitutes a possible persistent marker of P. brasiliensis infection.     

Proteomics-based identification of HSP60 as a tumor-associated antigen in early stage breast cancer and ductal carcinoma in situ

The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumor-associated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) ( n = 49) or early stage breast cancer ( n = 58), other cancers ( n = 20), autoimmune disease ( n = 20), and healthy subjects ( n = 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression.     

Protein microarray signature of autoantibody biomarkers for the early detection of breast cancer

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Identification of ribosomal protein autoantigens

Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.     

Autoimmunity as a Candidate for the Etiopathogenesis of Meniere's Disease: Detection of Autoimmune Reactions and Diagnostic Biomarker Candidate

Meniere''s disease is an inner ear disorder that can manifest as fluctuating vertigo, sensorineural hearing loss, tinnitus, and aural fullness. However, the pathologic mechanism of Meniere''s disease is still unclear. In this study, we evaluated autoimmunity as a potential cause of Meniere''s disease. In addition we tried to find useful biomarker candidates for diagnosis. We investigated the protein composition of human inner ear fluid using liquid column mass spectrometry, the autoimmune reaction between circulating autoantibodies in patient serum and multiple antigens using the Protoarray system, the immune reaction between patient serum and mouse inner ear tissues using western blot analysis. Nine proteins, including immunoglobulin and its variants and interferon regulatory factor 7, were found only in the inner ear fluid of patients with Meniere''s disease. Enhanced immune reactions with 18 candidate antigens were detected in patients with Meniere''s disease in Protoarray analysis; levels of 8 of these antigens were more than 10-fold higher in patients than in controls. Antigen-antibody reactions between mouse inner ear proteins with molecular weights of 23–48 kDa and 63–75 kDa and patient sera were detected in 8 patients. These findings suggest that autoimmunity could be one of the pathologic mechanisms behind Meniere''s disease. Multiple autoantibodies and antigens may be involved in the autoimmune reaction. Specific antigens that caused immune reactions with patient''s serum in Protoarray analysis can be candidates for the diagnostic biomarkers of Meniere''s disease.     

Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting

Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.     

Identification of kinectin as a novel Behçet's disease autoantigen

There has been some evidence that Beh?et's disease (BD) has a significant autoimmune component but the molecular identity of putative autoantigens has not been well characterized. In the initial analysis of the autoantibody profile in 39 Chinese BD patients, autoantibodies to cellular proteins were uncovered in 23% as determined by immunoblotting. We have now identified one of the major autoantibody specificities using expression cloning. Serum from a BD patient was used as a probe to immunoscreen a lambdaZAP expression cDNA library. Candidate autoantigen cDNAs were characterized by direct nucleotide sequencing and their expressed products were examined for reactivity to the entire panel of BD sera using immunoprecipitation. Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sj?gren's syndrome. Six independent candidate clones were isolated from the cDNA library screen and were identified as overlapping partial human kinectin cDNAs. The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%) BD patient sera by immunoprecipitation of the in vitro translation products. Sera from controls showed no reactivity. The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed.     

Paracoccidioides lutzii Plp43 Is an Active Glucanase with Partial Antigenic Identity with P. brasiliensis gp43

Background

Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii (“Pb01-like”) apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43.

Methodology

We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients'' sera from Southwestern and Midwestern Brazil.

Principal Findings

We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species.

Conclusion

Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.