Development of High Affinity Calcitonin Analog Fragments Targeting Extracellular Domains of Calcitonin Family Receptors

The calcitonin and amylin receptors (CTR and AMY receptors) are the drug targets for osteoporosis and diabetes treatment, respectively. Salmon calcitonin (sCT) and pramlintide were developed as peptide drugs that activate these receptors. However, next-generation drugs with improved receptor binding profiles are desirable for more effective pharmacotherapy. The extracellular domain (ECD) of CTR was reported as the critical binding site for the C-terminal half of sCT. For the screening of high-affinity sCT analog fragments, purified CTR ECD was used for fluorescence polarization/anisotropy peptide binding assay. When three mutations (N26D, S29P, and P32HYP) were introduced to the sCT(22–32) fragment, sCT(22–32) affinity for the CTR ECD was increased by 21-fold. CTR was reported to form a complex with receptor activity-modifying protein (RAMP), and the CTR:RAMP complexes function as amylin receptors with increased binding for the peptide hormone amylin. All three types of functional AMY receptor ECDs were prepared and tested for the binding of the mutated sCT(22–32). Interestingly, the mutated sCT(22–32) also retained its high affinity for all three types of the AMY receptor ECDs. In summary, the mutated sCT(22–32) showing high affinity for CTR and AMY receptor ECDs could be considered for developing the next-generation peptide agonists.

Binding Specificity of the Lantibiotic-Binding Immunity Protein NukH

ABSTRACT NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to nukH-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin4-27 and T24A nukacin4-27, respectively) to nukH-expressing cells were considerably lower than those of nukacin4-27, suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin4-27, a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of nukH-expressing cells.

Detection of Varicella Zoster Virus Specific CD8 T Cells in Patients after T Cell Depleted Allogeneic Stem Cell Transplantation by a Novel Epitope Screening Technology.

The incidence of Varicella Zoster Virus (VZV) reactivation after T cell depleted allogeneic stem cell transplantation (TCD alloSCT) is 40% the first year after transplantation. CD8 T cells are important in herpes virus reactivations. To analyze the role of CD8 T cells in VZV reactivation, we set out to develop multimeric MHC peptide complexes to be able to identify VZV specific CD8 T cells. Predictive algorithms were used to generate potential immunogenic peptides from Immediate Early (IE) proteins 4, 62 and 63 of VZV that bind in HLA-A2. 127 nonamer peptides were synthesized and tested by a high throughput peptide binding assay (REVEAL5™ assay). Of the peptides that were capable of sufficient binding to the MHC molecules, ProVE™ Pentamers were synthesized. 61 Pentamers were produced and used to screen for HLA-A2 restricted VZV specific T cells in peripheral blood derived from patients with clinical VZV reactivation after TCD alloSCT. In 6 out of 18 HLA-A2 positive patients after VZV reactivation, at a median of 43 days after onset of reactivation, T cells stained positive with the multimeric MHC complex that binds the ALW peptide of the IE62 protein of VZV (IE62-ALW-A2). The percentage of IE62-ALW-A2 positive T cells detected in these 6 patients ranged from 0.02% – 0.13% of CD8 T cells, with a median of 0.04% of CD8 T cells. To confirm the specificity of the Pentamer positive T cells, the IE62-ALW-A2 Pentamer positive T cells were sorted from one patient single cell per well, and expanded. The T cell clones exerted cytolytic activity against HLA-A2 positive EBV-lymphoblastoid cell lines loaded with the IE62-ALW-A2 peptide, whereas unloaded target cells were not lysed. HLA-A2 positive COS cells transfected with the IE62 gene and thus endogenously expressing the IE62 protein were recognized by IE62-ALW-A2 positive T cells as demonstrated by IFNg production. In addition, peripheral blood lymphocytes were stimulated in vitro with the IE62-ALW-A2 peptide in combination with IL-2 and IL-15, and we demonstrated with this procedure that IE62-ALW-A2 positive CD8 T cells were present at a low frequency in another 3 of the 12 IE62-ALW-A2 negative patients. To study whether the immune response against the IE62-ALW-A2 epitope correlated with the course of the clinical infection, we studied the percentage of IE62-ALW-A2 positive T cell during a VZV reactivation in one patient. Six days prior to reactivation, only 0.03% of the CD8 T cells were IE62-ALW-A2 positive. At 42 days after onset of the reactivation, 0.23% of the CD8 T cells were Pentamer positive. After the reactivation resolved, IE-62-ALW-A2 specific CD8 T cells declined to 0.09% at day 49 and 0.03% at day 145 after reactivation. In conclusion, we identified by using a high throughput peptide binding assay and Pentamer production, a new immune dominant epitope of VZV that is recognized by HLA-A2 restricted CD8+ T cells. We demonstrate that IE62-ALW-A2 specific T cells can recognize and lyse target cells that endogenously express the IE62 protein of VZV. In addition, we demonstrate that IE62-ALW-A2 specific T cells expand during the VZV specific immune response. This dominant epitope of VZV in combination with multimeric complexes can be a useful tool to study the immune response to VZV infection and possibly to VZV vaccination.

The Retrieval Function of the KDEL Receptor Requires PKA Phosphorylation of Its C-Terminus

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.

Induction of M3-Restricted Cytotoxic T Lymphocyte Responses by N-Formylated Peptides Derived from Mycobacterium tuberculosis

Major histocompatibility complex (MHC) class I–restricted CD8+ T cells play a critical role in the protective immunity against Mycobacterium tuberculosis (Mtb). However, only a few Mtb peptides recognized by MHC class Ia–restricted CD8+ T cells have been identified. Information on epitopes recognized by class Ib–restricted T cells is even more limited. M3 is an MHC class Ib molecule that preferentially presents N-formylated peptides to CD8+ T cells. Because bacteria initiate protein synthesis with N-formyl methionine, the unique binding specificity of M3 makes it especially suitable for presenting these particular bacterial epitopes. We have scanned the full sequence of the Mtb genome for NH2-terminal peptides that share features with other M3-binding peptides. Synthetic peptides corresponding to these sequences were tested for their ability to bind to M3 in an immunofluorescence-based peptide-binding assay. Four of the N-formylated Mtb peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes. The Mtb peptide–specific, M3-restricted CTLs lysed the Mtb-infected macrophages effectively, suggesting that these N-formylated Mtb peptides are presented as the naturally processed epitopes by Mtb-infected cells. Furthermore, T cells from Mtb-infected lungs, spleen, and lymph nodes responded to N-formylated Mtb peptides in an M3-restricted manner. Taken together, our data suggest that M3-restricted T cells may participate in the immune response to Mtb.