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Abstract We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC 50 ) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright , Half-Roll , and Roll . Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll , was constructed and named PharmaLogical . A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening.

Peptide-based drugs are an attractive new modality of therapeutics, and in vitro selection from a large-scale library is a powerful way to identify new lead sequences. In conventional screenings, peptide specificity and stability in physiological heterogenous environments are not evaluated, which sometimes makes subsequent optimization difficult. Here we show that selection using a cDNA display system can be performed in a high percentage of serum and that this might be an option to select molecules with high potency and stability in a biological context. Specifically, we chose interleukin-17A as a target protein and performed in vitro selection of cyclic peptide aptamers from a library of approximately 10 12 members in the presence of serum. The selected molecules had nanomolar affinity to the target and were stable in serum. Interestingly, we found that a component of the DNA linker that connected the peptide and cDNA may play a pivotal role in target binding., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

A massive deletion over three exons 16-17b in the CFTR gene was identified in Japanese CF patients with the highest frequency (about 70% of Japanese CF patients definitely diagnosed). This pathogenic mutation results in a deletion of 153 amino acids from glycine at position 970 (G970) to threonine at 1122 (T1122) in the CFTR protein without a frameshift. We name it Δ(G970-T1122)-CFTR. In the present study, we characterized the Δ(G970-T1122)-CFTR expressed in CHO cells using immunoblots and a super resolution microscopy. Δ(G970-T1122)-CFTR proteins were synthesized and core-glycosylated but not complex-glycosylated. This observation suggests that the Δ(G970-T1122) mutation can be categorized into the class II mutation like ΔF508. However, VX-809 a CFTR corrector that can help maturation of ΔF508, had no effect on Δ(G970-T1122). Interestingly C-terminal FLAG tag seems to partially rescue the trafficking defect of Δ(G970-T1122)-CFTR; however the rescued Δ(G970-T1122)-CFTR proteins do not assume channel function. Japanese, and perhaps people in other Asian nations, carry a class II mutation Δ(G970-T1122) with a higher frequency than previously appreciated. Further study of the Δ(G970-T1122)-CFTR is essential for understanding CF and CFTR-related diseases particularly in Asian countries.

Background: XIAP-associated factor 1 (XAF1) is ubiquitously expressed in normal tissues, but its suppression in cancer cells is strongly associated with tumor progression. Although downregulation of XAF1 is observed in tumors, its expression profile in the peripheral blood of cancer patients has not yet been investigated. Here, we identified a novel XAF1 splice variant in cancer cells and then investigated the expression level of this variant in peripheral blood containing gastric cancer-derived circulating tumor cells (CTCs)., Methods: To identify splice variants, RT-PCR and DNA sequencing were performed in mRNAs extracted from many cancer cells. We then carried out quantitative RT-PCR to investigate expression in peripheral blood from all 96 gastric cancer patients and 22 healthy volunteers., Results: The XAF1 variant harbored a premature termination codon (PTC) and was differentially expressed in highly metastatic cancer cells versus the parental cells, and that nonsense-mediated mRNA decay (NMD) was suppressed in the variant-expressing cells. Furthermore, splice variants of XAF1 were upregulated in peripheral blood containing CTCs. In XAF1 variant-expressing patients, the expression levels of other NMD-targeted genes also increased, suggesting that the NMD pathway was suppressed in CTCs., Conclusions: Our study identified a novel splice variant of XAF1 in cancer cells. This variant was regulated through the NMD pathway and accumulated in NMD-suppressed metastatic cancer cells and peripheral blood containing CTCs. The presence of XAF1 transcripts harboring the PTC in the peripheral blood may be useful as an indicator of NMD inhibition in CTCs.

Carcinoembryonic antigen (CEA), an oncofetal cell surface glycoprotein, has been widely used as a human tumor marker due to its high expression in tumors and secretion to serum. It belongs to the immunoglobulin superfamily named CEA-related cell adhesion molecule (CEACAM) family. Members of this family are detected in various cancers and have been shown to be involved in cancer growth and invasion. In this study, we examined the mRNA expression profiles of CEACAM family members including CEACAM1, CEACAM3, CEACAM4, CEACAM5 (CEA), CEACAM6, CEACAM7, and CEACAM8 in various tumor cell lines. Our screening data indicated that the mRNA expression patterns of CEACAMs in TT cells, which are derived from medullary thyroid carcinoma (MTC), were distinct from other tumor cell lines. Additionally, CEACAM4 was only expressed in TT cells, in which two novel splice variants of CEACAM4 were expressed. These findings suggested that production of CEA and CEA-related molecules in MTC may be distinct from other tumor-based production of those molecules and that the specific expression of CEACAM4 would make possible to differentiate between MTC and other CEA-producing tumors.

Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines., Results: We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns., Conclusions: This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker.

The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP/MXR/ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation.