Amnart Khongmanee

Here we describe a novel catalyst‐free 1,3‐dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)‐containing protein reacts with nitrile oxides generated in situ through 1,3‐dipolar cycloaddition in fully aqueous‐buffered systems. This leads to the formation of a new isoxazoline ring at a pre‐defined site (Dha) of the protein. Furthermore, the 1‐pyrene isoxazoline‐installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA‐1) cells for detection of apoptosis.

Programmed cell death protein 1 (PD-1) is a key receptor in the immune checkpoint pathway and has emerged to be a promising target for cancer therapy. PD-1 consists of an intracellular domain followed by a transmembrane domain that is connected to the extracellular domain by the stalk region. Although the PD-1 structure has been studied for more than two decades, the posttranslational modification of this protein has been incompletely characterized. In this study, we identified the previously undescribed modification sites of O-linked glycan on the stalk region of PD-1 protein using O-protease digestion coupling with intact mass analysis. The result indicates that T153, S157, S159, and T168 are modified by sialylated mucin-type O-glycan with core 1- and core 2-based structures. This study provides both information on potential novel modification sites on the PD-1 protein and an attractive method for identifying O-linked glycosylation using a specific enzyme and intact mass analysis.

Programmed cell death protein 1 (PD-1) is a key receptor in the immune checkpoint pathway and has emerged to be a promising target for cancer therapy. PD-1 consists of an intracellular domain followed by a transmembrane domain that is connected to the extracellular domain by the stalk region. Although the PD-1 structure has been studied for more than two decades, the posttranslational modification of this protein has been incompletely characterized. In this study, we identified the previously undescribed modification sites of O-linked glycan on the stalk region of PD-1 protein using O-protease digestion coupling with intact mass analysis. The result indicates that T153, S157, S159, and T168 are modified by sialylated mucin-type O-glycan with core 1–and core 2–based structures. This study provides both information on potential novel modification sites on the PD-1 protein and an attractive method for identifying O-linked glycosylation using a specific enzyme and intact mass analysis.

Colorectal cancer (CRC) is a major cause of cancer mortality. Currently used CRC biomarkers provide insufficient sensitivity and specificity; therefore, novel biomarkers are needed to improve the CRC detection. Label-free quantitative proteomics were used to identify and compare glycoproteins, enriched by wheat germ agglutinin, from plasma of CRC patients and age-matched healthy controls. Among 189 identified glycoproteins, the levels of 7 and 15 glycoproteins were significantly altered in the non-metastatic and metastatic CRC groups, respectively. Protein-protein interaction analysis revealed that they were predominantly involved in immune responses, complement pathways, wound healing and coagulation. Of these, the levels of complement C9 (C9) was increased and fibronectin (FN1) was decreased in both CRC states in comparison to those of the healthy controls. Moreover, their levels detected by immunoblotting were validated in another independent cohort and the results were consistent with in the study cohort. Combination of CEA, a commercial CRC biomarker, with C9 and FN1 showed better diagnostic performance. Interestingly, predominant glycoforms associated with acetylneuraminic acid were obviously detected in alpha-2 macroglobulin, haptoglobin, alpha-1-acid glycoprotein 1, and complement C4-A of CRC patient groups. This glycoproteomic approach provides invaluable information of plasma proteome profiles of CRC patients and identification of CRC biomarker candidates.

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Cholangiocarcinoma (CCA), a high‐prevalence cancer in Thailand, is highly metastatic and has high mortality rates. Anoikis resistance or the ability of cells to survive after detachment from extracellular matrix is a necessary property of metastatic cancer. Here, we report differential protein expression of an anoikis‐resistant CCA cell line culture, under attachment conditions compared to nonattachment conditions, studied using 2DE coupled with protein identification by LC‐MS/MS. Our data reveal the deregulation of proteins involved in stress response, cytoskeleton rearrangement, proapoptosis, cell proliferation, and glycolysis. Interestingly, 14‐3‐3σ (14‐3‐3 sigma) protein was intensely upregulated in detached CCA cells. Real‐time RT‐PCR analysis confirmed that only the sigma isotype was the most abundant transcript among 14‐3‐3 genes in CCA cells. Furthermore, silencing 14‐3‐3σ expression by small interfering RNA in CCA cells resulted in significantly increased percentage of cell death in detached culture. Our findings provide the first evidence showing that 14‐3‐3σ protein plays a crucial role in anoikis resistance of CCA cells. Therefore, 14‐3‐3σ might be a potential target in CCA therapy.

Background and Objectives: Sprouty (Spry) proteins are important modulators of the RTK/Ras/MAPK pathway, overactivation of which is associated with hepatocellular carcinoma (HCC). Thus far, the roles of Sprouty in HCC is still unclear.Methods: The expressions of SPRY1, SPRY2, SPRY3, and SPRY4, at the mRNA levels were determined by quantitative RT‐PCR in paired HCC and non‐tumor liver tissues from 31 patients.Results: The expression levels of SPRY1, SPRY2, and SPRY4 in tumor tissues were significantly different from those in non‐tumor tissues with the average log fold change values of 0.15, −0.34, and −0.37, respectively; however, that of SPRY3 was not significantly different. SPRY1 expression was also found to be significantly up‐regulated in the cases without underlying cirrhosis compared with those with cirrhosis (log fold change of 0.35 and −0.02, respectively, P < 0.05), whereas SPRY2 expression was significantly lower in the cases with advanced HCC (log fold change of −0.12 and −0.52 in early and advanced stages, respectively, P < 0.05) and in those with angiolymphatic invasion (log fold change of −0.47 and −0.16 in the presence and absence thereof, P < 0.05).Conclusion: This study demonstrates that Sprouty genes are differentially expressed in HCC and might provide some insight into their roles in HCC carcinogenesis. J. Surg. Oncol. 2012; 105:273–276.