The role of N-linked oligosaccharide structure in regulation of immunoglobulin secretion and function

The functions of carbohydrate moieties of glycoproteins are subject to much controversy. Their role in the regulation of intracellular glycoprotein transport is obscured by the discovery of distinctive effects that result from treatment either with inhibitors of N-glycosylation or intracellular N-linked oligosaccharide processing. It is therefore the purpose of this study to ratify and to broaden the perspective in this area of ambiguity. The experimental approach used in the investigation generally involves biosynthetic labelling of rat hybridoma cells with various radioactive precursor and an isolation of intracellular and/or extracellular immunoglobulins by immunoprecipitation technique. Through properly-timed, pulse-chase methods, it was possible to determine the effects of various inhibitors of glycosylation or oligosaccharide processing on the kinetics of immunoglobulin export. The isolated immunoglobulins were also subjected to other analyses to identify and to confirm the structures of carbohydrate molecules that were incorporated, as well as to assess the effects of inhibition of N-glycosylation or N-linked oligosaccharide processing on the biological functions of immunoglobulins. The first part of this study involves characterization of three structural analogues of tunicamycin, TM-1, TM-2 and TM-3. Results from this investigation demonstrate that minor modifications to the structure of the antibiotic results in the loss of its biological activity (i.e., with respect to inhibition of N-glycosylation). The analogues do not inhibit N-glycosylation of immunoglobulins and have no effects on the kinetics of IgM and IgG2b secretion from rat hybridomas. The data, therefore, suggest that the selective inhibition of immunoglobulin secretion that is observed when cells are treated with tunicamycin is really due to the absence of carbohydrate moieties from the immune molecules and not a direct effect of the antibiotic. The data also demonstrate, that unlike normal tunicamycin, the three structural analogues are not cytotoxic. Their presence has no pronounced effect on the cellular uptake of tritiated-thymidine, -uridine and -leucine. This suggests that the well-recognized cytotoxic effects of tunicamycin are also a secondary effect due to the action of the drug on other cellular functions. In addition, enzymatic analysis of isolated immunoglobulins produced and secreted in the presence of the three tunicamycin analogues demonstrates that the drugs have no effect on the N-acetylglucosaminyl transferase I and II activities located in the Golgi apparatus. Susceptibility to endo H digestion is only observed in immunoglobulins isolated from within the cells. Immunoglobulins secreted in the presence of the analogues of tunicamycin display complete resistance to the enzyme. To provide a wider perspective on the current understanding of the role of N-linked carbohydrate processing in the regulation of glycoprotein transport, a study of the effects of four specific N-linked oligosaccharide processing inhibitors (i.e., CSP, dNM, dMM and SW) on the secretion of IgM and IgG2b from rat hybridoma lines was carried out. The data clearly demonstrate that inhibition of the processing of N-linked oligosaccharide at specific stages of the pathway does not lead to any significant interference with the rate of IgM and IgG2b secretion. Resolution of the reduced immunoglobulin components secreted from cells that were treated with the individual processing inhibitors on SDS-PAGE demonstrate distinctive heavy chain structures. While no apparent difference could be seen from the mu- and gamma-heavy chains that were secreted in the presence of the mannosidase Ia/b and II inhibitors (i.e., dMM and SW, respectively), treatment with glucosidase inhibitors (i.e., CSP and dNM) results in the production of mu- and gamma-heavy chains with higher Mr. Experiments were also performed by using mixtures of the carbohydrate processing inhibitors. Treatment of I1A 1.4 and 4A3 cells with selective pairs of glucosidase and mannosidase inhibitors (i.e., CSP/SW and dNM/dMM) or with all the four processing inhibitors simultaneously, also demonstrate no significant changes of the rate of immunoglobulin export. The mu- and gamma-heavy chains that were isolated in all of these cases, however, demonstrate the higher-type Mr structures. The oligosaccharide chains of IgG2b from I1A1.4 cells, under normal circumstances, although bearing the complex-type structures, do not possess terminally-linked sialic acid residues. By using neuraminidase digestion analysis, it was shown that when the processing of the carbohydrate moieties was inhibited by any of the four processing inhibitors, individually, or with all of them simultaneously, g-heavy chains become susceptible to the enzyme treatment, suggesting the presence of terminally-linked sialic acid residues. This effect may either be a result of a direct activation of intracellular sialyl transferase or due to other processing resulting in carbohydrate configurations which can act as substrates for the transferase. N-linked carbohydrate moieties of immunoglobulins have been strongly implicated to be involved in the Clq-binding interaction. Their absence from immunoglobulins have rendered the molecules less effective in activating the complement cascade. The data from experiments performed in this study are also compatible with this interpretation. A similar result was obtained when tunicamycin-treated non-N-glycosylated IgG2b from I1A 1.4 cells was subjected to complement fixation assay. In the case of immunoglobulins with high mannose structures that were secreted in the presence of oligosaccharide processing inhibitors, the data indicate otherwise. These immunoglobulins apparently possess potentiated capability in fixing complement. Complement fixation assays were also performed on normal IgG2b in the presence of free mannose, galactose and N-acetylglucosamine at 5mM concentration. The monosaccharides have no effect on the complement fixation activity. The effects of inhibition of N-glycosylation or oligosaccharide processing on Fc-binding interactions, which may involve carbohydrate moieties, have also been studied. The data indicate that neither the inhibition of N-glycosylation nor carbohydrate processing have any consequence on antibody-dependent haemagglutination by IgG2b from I1A1.4 cells. In addition, inhibition of N-glycosylation or the processing of carbohydrate moieties of IgG2b was also shown to have no effect on antigen binding capacity of the immunoglobulins

Overview of Proteomics

Abstract The era of genomics has brought tremendous advancement especially in the fields of medicine and life sciences. Despite the overwhelming growth of information generated from genomics research, a huge gap specifically in relation to genome expression persists, and became increasingly noticeable. This has sparked interest in studies of proteins expression and eventually added limelight to the field of proteomics. Aside from the need to fill the gap, the emergence of proteomics is also deemed to have occurred due to the advancement in capabilities of research techniques, particularly in the separation and identification of proteins. Proteomics has since progressed and is slowly extending into other research as well as applied subspecialties. This brief overview was written to provide a basic and simplified understanding of proteomics in view of its growing interest from newcomers to the area

Patients with ovarian carcinoma excrete different altered levels of urine CD59, kininogen-1 and fragments of inter-alpha-trypsin inhibitor heavy chain H4 and albumin

CD59, kininogen-1 and fragments of ITIH4 and albumin may be used as complementary biomarkers in the development of new noninvasive protocols for diagnosis and screening of ovarian carcinoma

Moderating Effects of Personality Traits On Online Learning Transition and Acceptance Among Culinary Arts Students

 Many Higher Education Institution (HEI) students had to make an immediate change to online learning from the conventional face-to-face mode due to the COVID-19 pandemic and Movement Control Order (MCO) imposed by the government. Learning practical courses such as Culinary Arts via online without application or practical work generated bigger challenges for HEIs. It was emphasised that Culinary Arts education depends predominantly on hands-on application and training. The purpose of this study is to investigate Culinary Arts program students’ acceptance of online learning methods (hands-on learning at home) and how the Big Five Personality Traits (BFPT) could have an impact on the relationship. A total of 234 responses from Culinary Arts based program students of six (6) HEIs in Malaysia were obtained and analysed using SPSS statistical software. Findings showed that students were able to accept the transition in learning from face-to-face to online learning. However, it was found that BFPT did not have a significant moderating impact on the relationship between Learning Transition and Online Learning Acceptance. The results could help HEIs in adapting to the new Learning Transition without compromising the quality of the graduates and the curriculum set by the institutions. In addition, the results of this study could enhance further investigations on Online Learning Acceptance to a wider scope and type of study programs

Application of SELDI-TOF in N-glycopeptides profiling of the urine from patients with endometrial, ovarian and cervical cancer

Purpose: Endometrial (ECa), ovarian (OCa) and cervical (CCa) cancers are among 10 of the most common cancers affecting women worldwide. Cancers are known to cause some proteins to be differentially glycosylated or aberrantly excreted in the urine, which can be used as biomarkers. Since ECa, OCa and CCa are difficult to diagnose at the early stage, the aim of the present study was to identify a panel of new biomarkers for early detection of the cancers using surface-enhanced laser desorption/ionization-time-of-flight (SELDI-TOF) technology. Identification of early biomarkers that are specific and efficient can increase the survival rate of the patients. Experimental design: Digested urinary proteins from patients with ECa, OCa and CCa were incubated on the champedak mannose-binding (CMB) lectin-immobilized PS10 chip. The lectin-captured glycopeptides were detected with SELDI-TOF mass spectrometry and followed by biomarker wizard analysis. Results: Peaks m/z 1201 and 1449 were detected as potential group discriminators. The peak m/z 1201 could distinguish OCa from CCa and ECa and its sensitivity and specificity were 100%. For m/z 1449, it was able to differentiate ECa from the other two types of cancer. Conclusions: The findings of this study suggest urinary glycopeptides m/z 1201 and 1449 may serve as potential biomarkers for the early detection of ECa, OCa and CCa, although this requires further extensive validation on clinically representative populations

Profiling of Burkholderia cepacia Secretome at Mid-Logarithmic and Early-Stationary Phases of Growth

BACKGROUND: Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD), flagellar hook-associated protein (FlgK), TonB-dependent siderophore (Fiu), Elongation factor G (FusA), phosphoglycerate kinase (Pgk) and sulfatase (AslA) which are known for their virulence. CONCLUSION/SIGNIFICANCE: Differences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to control B. cepacia infection using agents that can block their release

Profiling of serum and tissue high abundance acute-phase proteins of patients with epithelial and germ line ovarian carcinoma

<p>Abstract</p> <p>Background</p> <p>Acute-phase response involves the simultaneous altered expression of serum proteins in association to inflammation, infection, injury or malignancy. Studies of the acute-phase response usually involve determination of the levels of individual acute-phase serum proteins. In the present study, the acute-phase response of patients with epithelial (EOCa) and germ-line (GOCa) ovarian carcinoma was investigated using the gel-based proteomic approach, a technique which allowed the simultaneous assessment of the levels of the acute-phase serum high abundance proteins. Data obtained were validated using ELISA and immunostaining of biopsy samples.</p> <p>Results</p> <p>Enhanced expression of clusterin (CLU), α₁-antitrypsin, haptoglobin and leucine rich glycoprotein was detected in all patients. However, the levels of α₁-antichymotrypsin (ACT) was only enhanced in EOCa patients, while patients with GOCa were typically characterized by elevated levels of ceruloplasmin but lower levels of α₂-HS glycoprotein. The enhanced expression of CLU in EOCa and GOCa patients and up-regulated expression of ACT specifically in EOCa patients were confirmed by ELISA. Immunohistochemical staining of biopsy samples of EOCa and GOCa patients demonstrated correlation of the acute-phase protein expression.</p> <p>Conclusion</p> <p>Patients with EOCa and GOCa demonstrated distinctive aberrant expression of serum and tissue high abundance acute-phase proteins compared to negative control women.</p

Characterization of significant molecular determinants of virulence of Enterovirus 71 sub-genotype B4 in Rhabdomyosarcoma cells

One of the leading causes of the hand, foot and mouth disease (HFMD) is Enterovirus 71(EV-A71), displaying symptoms such as fever and ulcers in children but some strains can produce cardiopulmonary oedema which leads to death. There is no FDA-approved vaccine for prevention of severe HFMD. The molecular determinants of virulence for EV-A71 are unclear. It could be a single or a combination of amino acids that determines virulence in different EV-A71 genotype/sub-genotypes. Several EV-A71 strains bearing single nucleotide (nt) mutations were constructed and the contribution of each mutation to virulence was evaluated. The nt(s) that contributed to significant reduction in virulence in vitro were selected and each mutation was introduced separately into the genome to construct the multiply mutated EV-A71 strain (MMS) which carried six substitutions of nt(s) at the 5’-NTR (U700C), VP1-145 (E to G), VP1-98E, VP1-244K and G64R in the vaccine seed strain that had a partial deletion within the 5’-NTR region (nt. 475-485) of ∆11bp. In comparison to the wild type strain, the MMS showed low virulence as it produced very low RNA copy number, plaque count, VP1 and had 105-fold higher TCID50, indicative of a promising LAV candidate that should be further evaluated in vivo

Papillary thyroid cancer: Genetic alterations and molecular biomarker investigations

Papillary thyroid cancer (PTC) is the most prevalent form of malignancy among all cancers of the thyroid. It is also one of the few cancers with a rapidly increasing incidence. PTC is usually contained within the thyroid gland and generally biologically indolent. Prognosis of the cancer is excellent, with less than 2% mortality at 5 years. However, more than 25% of patients with PTC developed a recurrence during a long term follow-up. The present article provides an updated condensed overview of PTC, which focuses mainly on the molecular alterations involved and recent biomarker investigations

Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats

<p>Abstract</p> <p>Background</p> <p>Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR).</p> <p>Results</p> <p>Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril.</p> <p>Conclusion</p> <p>Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension.</p