Phannee Sawangareetrakul

Proteomic studies of cholangiocarcinoma and hepatocellular carcinoma cell secretomes.

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1) and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander) cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2), laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

Novel mutations in a Thai patient with methylmalonic acidemia

A Thai patient with methylmalonic acidemia (MMA) and no methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) activity in leukocytes in the presence of deoxyadenosyl cobalamin (mut(0)) was found to be heterozygous for two novel mutations: 1048delT and 1706_1707delGGinsTA (G544X), inherited from her mother and father, respectively. The proband was also heterozygous for the polymorphism, A499T, which did not affect the activity of recombinant MCM.

Clinical and molecular findings in Thai patients with isolated methylmalonic acidemia

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous organic acid disorder caused by either deficiency of the enzyme methylmalonyl-CoA mutase (MCM), or a defect in the biosynthesis of its cofactor, adenosyl-cobalamin (AdoCbl). Herein, we report and review the genotypes and phenotypes of 14 Thai patients with isolated MMA. Between 1997 and 2011, we identified 6 mut patients, 2 cblA patients, and 6 cblB patients. The mut and cblB patients had relatively severe phenotypes compared to relatively mild phenotypes of the cblA patients. The MUT and MMAB genotypes were also correlated to the severity of the phenotypes. Three mutations in the MUT gene: c.788G>T (p.G263V), c.809_812dupGGGC (p.D272Gfs*2), and c.1426C>T (p.Q476*); one mutation in the MMAA gene: c.292A>G (p.R98G); and three mutations in the MMAB gene: c.682delG (p.A228Pfs*2), c.435delC (p.F145Lfs*69), and c.585-1G>A, have not been previously reported. RT-PCR analysis of a common intron 6 polymorphism (c.520-159C>T) of the MMAB gene revealed that it correlates to deep intronic exonization leading to premature termination of the open reading frame. This could decrease the ATP:cobalamin adenosyltransferase (ATR) activity resulting in abnormal phenotypes if found in a compound heterozygous state with a null mutation. We confirm the genotype-phenotype correlation of isolated MMA in the study population, and identified a new molecular basis of the cblB disorder.

Comparative secretome analysis of cholangiocarcinoma cell line in three-dimensional culture

Cholangiocarcinoma (CCA) is a lethal malignancy which occurs with relatively high incidence in Thailand. This cancer is often difficult to diagnose and associated with high mortality. The secretome, containing the secreted proteins from cells, are potentially useful as biomarkers of cancers. Since three-dimensional (3D) cell culture may mimic growth characteristics and microenvironment of solid tumors in vivo better than monolayer culture, we have developed culture of CCA in natural collagen-based scaffold, to enable analysis of the secretome by 2DE. Our results indicated that CCA growth in 3D environment alters cell shape significantly and enhances extracellular matrix deposition. Interestingly, more secreted proteins were detected from 3D culture compared to monolayer culture. Secretome analysis using 2DE coupled with LC-MS/MS demonstrated 10 secreted proteins uniquely found in 3D culture. Moreover, 25 proteins were enriched in 3D culture compared to monolayer culture, including 14-3-3 σ, triosephosphate isomerase, phosphoglycerate mutase 1, α-enolase, and L-plastin. Immunoblotting was used to confirm the presence of L-plastin in conditioned media of CCA and of hepatocellular carcinoma (HCC) cell lines. The results revealed that L-plastin, an actin bundling protein, was uniquely expressed only in the CCA cell line and could be a promising biomarker for differential diagnosis of CCA compared to HCC.

Comparison of membrane-associated proteins in human cholangiocarcinoma and hepatocellular carcinoma cell lines

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. Cell line models, originating from Thai patients, are available for both diseases, including the human bile duct epithelial carcinoma cell line (HuCCA‐1) and the HCC cell line HCC‐S102. Here, we have prepared subproteomes enriched in membrane proteins or in cytosolic proteins from the HuCCA‐1 and the HCC‐S102 cell lines. Study of differential protein expression by 2‐DE and LC/MS/MS showed 195 proteins expressed in the two cell lines, including both membrane‐associated and cytosolic proteins. Eighteen proteins were found in both membrane and cytosolic fractions of HuCCA‐1, but not in HCC‐S102, while nine proteins were found in both membrane and cytosolic fractions of HCC‐S102, but not in HuCCA‐1. Ten membrane proteins were found in HuCCA‐1 but not in HCC‐S102, including integrin alpha‐6 precursor, ezrin, hippocalcin‐like protein 1, mitogen‐activated protein kinase kinase kinase 2 (MAPK/ERK kinase kinase 2), and calgizzarin. Proteins showing increased expression in the membrane fraction of HuCCA‐1 were mainly cytoskeletal proteins (40.9%), while proteins showing increased expression in the membrane fraction of HCC‐S102 were mainly metabolic proteins (39.4%). The subproteomic approach used here facilitates detection of potential biomarkers undetected by regular proteomic methods.

Decreasing activity and altered protein processing of human iduronate-2-sulfatase mutations demonstrated by expression in COS7 cells.

Hunter syndrome, or mucopolysaccharidosis type II (MPS II; MIM:309900), is a rare X-linked recessive lysosomal storage disorder caused by defects of the gene coding for iduronate-2-sulfatase (IDS; EC 3.1.6.13) (Bach et al. 1973). IDS catabolizes dermatan sulfate and heparan sulfate in the lysosome. Lack of this enzyme leads to the systemic accumulation of these substances, eventually causing characteristic clinical

ASSOCIATION OF Hb HOPE [β136(H14)Gly → Asp] AND Hb H DISEASE

Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, 25=25 Phuttamonthon 4 Road, Phuttamonthon, Nakornpathom 73170, Thailand Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Hematology Section, Departments of Pathology and Medicine, Rajavithi Hospital, Bangkok 10400, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand Institute of Science, Suranaree University of Technology, Nakorn Ratchasima 30000, Thailand

Isolated methylmalonic acidemia with unusual presentation mimicking diabetic ketoacidosis.

Abstract Background: Hyperglycemic ketoacidosis is an acute, life threatening condition requiring early etiologic recognition and management to prevent serious morbidity/mortality. The most common cause is diabetic ketoacidosis (DKA). Organic acidemias (OAs) are inheritable disorders caused by defects in protein metabolism resulting in acid accumulation. Patients with metabolic decompensation usually present with acidosis, with/without hypoglycemia. Hyperglycemia is a very rare manifestation. At least 16 cases of OAs presenting with hyperglycemia have been reported. Six of the 16 were diagnosed with isolated methylmalonic academia (MMA) and three of the six passed away from late diagnosis. Case description: We describe a 2-year-old Thai girl who presented with hyperglycemia, acidosis and ketosis. She has underlying delayed development, seizures, optic atrophy and poor growth. An initial diagnosis of DKA was made and standard treatment was started. After 4 h of treatment, the patient partially responded to treatment; blood sugar decreased but acidosis and ketonemia persisted. HbA1c was normal. Investigations to rule out OAs were performed. Markedly elevated urinary methylmalonic acid consistent with MMA was observed. Molecular and enzyme analyses confirmed the diagnosis with isolated MMA. Specific treatment for MMA including protein restriction, high caloric fluid, carnitine and vitamin B12 was promptly started. Clinical improvement was seen 4 days after initiating specific treatment. Conclusions: Inherited metabolic disorders should be included in differential diagnosis in hyperglycemia ketoacidosis patients who respond poorly to standard DKA treatment. Unusual findings, e.g. hyperammonemia, lactic acidosis, pancytopenia, abnormal basal ganglia in MRI or underlying delayed development may indicate underlying OAs. Determining the etiology of hyperglycemic ketoacidosis is important and can lead to good outcomes.

β-Elimination coupled with strong cation-exchange chromatography for phosphopeptide analysis

RATIONALE Since the last decade, mass spectrometry (MS) has become an essential technique for phosphoprotein analysis. Formidable analytical challenges of MS for phosphoprotein study are both the low abundance of phosphopeptides and the lack of an unambiguous diagnostic fragment ion for identification of phospho residues. These challenges can be met by a charge-based isolation of β-elimination products after tryptic digestion using diagonal strong cation-exchange chromatography. METHODS β-Elimination combined with diagonal strong cation-exchange chromatography (BE/2SCX) was used for the enrichment of phosphorylated peptides prior to a mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (MS/MS). Bovine α-casein (≥70% purity) was used as a model protein. RESULTS Conditions for β-elimination were optimized to maximize the efficiency of the reaction. With a β-elimination, all four model phosphopeptides from enolase (yeast) were correctly identified. The application of the BE/2SCX enrichment strategy for the analysis of β-elimination products of α-casein (bovine) allowed the identification of 11 phosphorylated products. CONCLUSIONS The introduction of a BE/2SCX-based enrichment step prior to LC/MS/MS analysis of β-elimination products facilitates the identification of phosphopeptides. Copyright

DOUBLE HETEROZYGOSITY FOR Hb PYRGOS [β83(EF7)Gly→Asp] AND Hb E [β26(B8)Glu→Lys] FOUND IN ASSOCIATION WITH α-THALASSEMIA

Thailand has a high prevalence of aand b-thalassemias and abnormal hemoglobins (Hbs), especially Hb E [b26(B8)Glu!Lys] and Hb Constant Spring (CS) [a142 (Term!Gln) (TAA!CAA in a2)]. As a result, interactions of various abnormal genes have been found, such as Hb C [b6(A3)Glu!Lys]= Hb E, Hb Siam (also known as Hb Ottawa) [a15(A13)Gly!Arg (a1)] with a-thalassemia (thal), and Hb Hope [b136(H14)Gly!Asp] with Hb H (b4) disease. In this paper we report two cases of double heterozygosity for Hb Pyrgos [b83(EF7)Gly!Asp] and Hb E, in association with a-thal.