Immuno-informatics analysis and expression of a novel multi-domain antigen as a vaccine candidate against glioblastoma.

Glioblastoma multiform is the most common of primary malignant brain tumors in adults. Currently, surgical resection of the tumor mass, followed by adjuvant radiotherapy and chemotherapy are standard treatments for glioblastoma multiform but so far are not effective treatments. Thus, the development of a vaccine, as a safe and efficient strategy for prophylactic or therapeutic purposes against glioblastoma multiform is very necessary. The present study aimed to design the multi-domain vaccine for glioblastoma multiform. An in silico approach was used to select the most potent domains of proteins to induce the host's B- and T-cell immune response against glioblastoma multiform. IL-13Rα-2 (amino acid positions 27-144), TNC (amino acid positions 1900-2100), and PTPRZ-1(amino acid positions 731-884) were found to have potent inducible immune responses. So, we considered them for fusing with a linker A(EAAAK)3A to construct the multi-domain recombinant vaccine. The immuno-informatics analysis of the designed recombinant vaccine construct was performed to evaluate its efficacy. Although the designed recombinant vaccine construct did not show allergen property, its antigenicity was estimated at 0.78. The Physico-chemical properties of the recombinant vaccine construct were characterized and revealed the potency of the vaccine candidate. Then its secondary and tertiary structures, mRNA structure, molecular docking, and immune simulation were predicted using bioinformatics tools. Next, the designed recombinant vaccine construct was synthesized, and cloned into the pET28a vector and expressed in E. coli BL21. Besides, the circular dichroism spectroscopy was utilized for the investigation of the secondary structure changes of the recombinant vaccine construct. The results of the verification assessment of the recombinant vaccine construct expression indicated that in silico analysis was relatively accurate, and relatively change occurred on the protein secondary structure. In our future plan, the vaccine candidate that was confirmed by in silico tools should be validated by further in vitro and in vivo experimental studies.

A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients.

BACKGROUND: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. METHODS: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. RESULTS: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. CONCLUSIONS: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions.

Protein tyrosine phosphatase receptor type γ is a JAK phosphatase and negatively regulates leukocyte integrin activation.

Regulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T lymphocytes. However, the role of protein tyrosine phosphatases in leukocyte recruitment is still elusive. In this study, we address this issue by focusing on protein tyrosine phosphatase receptor type γ (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine phosphatases and found that activated PTPRG blocks chemoattractant-induced ß2 integrin activation. Specifically, triggering of LFA-1 to high-affinity state is prevented by PTPRG activation. High-throughput phosphoproteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 dephosphorylation on the critical 1007-1008 phosphotyrosine residues, implying JAK2 inhibition and thus explaining the antiadhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG downmodulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical regulator of leukocyte trafficking.

Binding specificity of anti-HNK-1 IgM M-protein in anti-MAG neuropathy: possible clinical relevance.

Anti-myelin-associated-glycoprotein (MAG) neuropathy is an intractable autoimmune polyneuropathy. The antigenic region of MAG is the human natural killer-1 (HNK-1) carbohydrate. We and others previously suggested that the extension of antibody reactivities to HNK-1-bearing proteins other than MAG was associated with treatment resistance, without statistical analyses. In this study, we established an ELISA method with recombinant proteins to test binding specificities of currently available monoclonal antibodies to MAG and another HNK-1-bearing protein, phosphacan. Using this system, we found the distinct binding specificities of anti-MAG antibody in 19 patients with anti-MAG neuropathy. Their clinical relevance was then determined retrospectively with the adjusted 10-points INCAT disability score (0 = normal and 10 = highly disable). The results showed that strong reactivities of anti-MAG antibodies to phosphacan were significantly associated with treatment resistance or progressive clinical courses, indicating a possible clinical relevance of the binding specificities.

Reduced expression of MAb6B4 epitopes on chondroitin sulfate proteoglycan aggrecan in perineuronal nets from cerebral cortices of SAMP10 mice: a model for age-dependent neurodegeneration.

The accelerated senescence-prone SAMP10 mouse strain is a model for age-dependent neurodegeneration and is characterized by brain atrophy and deficits in learning and memory. Because perineuronal nets play an important role in the synaptic plasticity of adult brains, we examined the distributions of molecules that constitute perineuronal nets in SAMP10 mouse brain samples and compared them with those in control SAMR1 mouse samples. Proteoglycan-related monoclonal antibody 6B4 (MAb6B4) clearly immunostained perineuronal nets in SAMR1 mice cortices, but the corresponding immunostaining in SAMP10 mice was very faint. MAb6B4 recognizes phosphacan/PTPzeta in immature brains. However, this antibody recognized several protein bands, including a 400-kDa core glycoprotein from chondroitin sulfate proteoglycan in homogenates of mature cortices from SAMR1 mice. The 400-kDa band was also recognized by antiaggrecan antibodies. The aggrecan core glycoprotein band was also detectable in samples from SAMP10 mice, but this glycoprotein was faintly immunostained by MAb6B4. Because MAb6B4 recognized the same set of protein bands that the monoclonal antibody Cat-315 recognized in mature cerebral cortices of SAMR1 mice, the MAb6B4 epitope appears to be closely related to that of Cat-315 and presumably represents a novel type of oligosaccharide that attaches to aggrecans. The Cat-315 epitope colocalized with aggrecan in perineuronal nets from SAMR1 mouse brain samples, whereas its expression was prominently reduced in SAMP10 mouse brain samples. The biological significance of the MAb6B4/Cat-315 epitope in brain function and its relationship to the neurodegeneration and learning disabilities observed in SAMP10 mice remain to be elucidated.