Synthesis of tools for glycoprotein remodelling

The production of therapeutic glycoproteins typically relies on the purification of the desired glycoprotein from engineered tissue culture systems. This process is costly and inefficient, and the isolated glycoproteins are adorned with an array of heterogeneous sugars. Expression of glycoproteins in cheaper non-mammalian host cells such as yeast produces large quantities of folded glycoproteins though the sugar chains are immunogenic to humans. This project initially aimed to develop a general strategy for the humanisation of yeast glycoproteins using the copper (I) mediated ligation between azide and acetylene functional groups, commonly known as “click” chemistry. It required that azide and acetylene groups could be efficiently incorporated into (glyco)peptides and sugars respectively, and to this end propargylic glycosides of mannose, N-acetyl glucosamine, and N-acetyl lactosamine were successfully prepared. Two different glycoprotein remodelling methods were ultimately investigated. The first method explored a chemoenzymatic strategy developed in our group. A target glycoprotein, erythropoietin (EPO), was expressed in the presence of azide bearing mannose sugars in the hope that azide groups would be incorporated during glycoprotein biosynthesis. Subsequent elaboration of the azide functionality with propargyl N-acetyl lactosamine followed by enzymatic sialylation was expected to yield a glycoprotein with human-like sialyl lactosamine antennae. However, upon purification from Pichia pastoris incorporation of azide could not be detected, limiting the strategy to in vitro glycoprotein remodelling. Several technological advances were made including optimisation of the final “click” reaction between the propargyl glycoside of N-acetyllactosamine and 4-azidomannose followed by enzymatic transfer of sialic acid. While considerable effort was directed towards the key transfer of unnatural azidosugars, from nucleotide donors to potential substrates using an α-1,2-mannosyltransferase, this step proved unreliable. The second method introduced azide groups into fully synthetic peptides using the amino acid azidohomoalanine (Aha). Click chemistry with synthetic propargyl glycosides allowed further modification to homogeneous glycopeptide analogues which were shown to be compatible with native chemical ligation, a proven tool for glycoprotein synthesis and semisynthesis. Ultimately both methods may eventually facilitate glycoprotein synthesis and remodelling such that the biological activity and immunogenicity may be modulated to suit future therapeutic requirements

The uremic toxin oxythiamine causes functional thiamine deficiency in end-stage renal disease by inhibiting transketolase activity

Decreased transketolase activity is an unexplained characteristic of patients with end stage renal disease (ESRD) and is linked to impaired metabolic and immune function. Herein we describe the discovery of a link to impaired functional activity of thiamine pyrophosphate co-factor through the presence, accumulation and pyrophosphorylation of the thiamine antimetabolite, oxythiamine, in renal failure. Plasma oxythiamine was increased 4-fold in patients receiving continuous ambulatory peritoneal dialysis (CAPD) and 15-fold in patients receiving haemodialysis (HD) immediately before a dialysis session: healthy controls 0.18 (0.11 – 0.22) nM, CAPD, 0.64 (0.48-0.94) nM and HD (2.73 (1.52-5.76) nM); P<0.001, Mann-Whitney U test. Oxythiamine was converted to the transketolase inhibitor oxythiamine pyrophosphate (OTPP). Red blood cell OTPP concentration was increased 4-fold in HD: healthy controls, 15.9 ± 10.4 nM and HD patients, 66.1 ± 26.7 nM; P<0.001, t-test. This accounted for the concomitant 41% loss of transketolase activity (mU/mg Hb): healthy controls, 0.410 ± 0.144 nM and HD, 0.240 ± 0.107 nM; P<0.01, paired t-test. This may be corrected by displacement with excess thiamine pyrophosphate and explain lifting of decreased transketolase activity by high dose thiamine supplementation in previous studies. Oxythiamine is likely of dietary origin, through cooking of acidic thiamine-containing foods. Trace level oxythiamine was not formed from thiamine degradation under physiological conditions but rather under acidic conditions at 100 oC. Monitoring of plasma oxythiamine concentration in renal failure and implementation of high dose thiamine supplements to counter it may help improve clinical outcome of patients with renal failure

Інтеграційні процеси в паливно-енергетичному комплексі як фактор забезпечення екологічної безпеки

Challenge tests stress homeostasis and may reveal deviations in health that remain masked under unchallenged conditions. Ideally, challenge tests are non-invasive and applicable in an early phase of an animal experiment. Oxygen restriction (OxR; based on ambient, mild normobaric hypoxia) is a non-invasive challenge test that measures the flexibility to adapt metabolism. Metabolic inflexibility is one of the hallmarks of the metabolic syndrome. To test whether OxR can be used to reveal early diet-induced health effects, we exposed mice to a low-fat (LF) or high-fat (HF) diet for only 5 days. The response to OxR was assessed by calorimetric measurements, followed by analysis of gene expression in liver and epididymal white adipose tissue (eWAT) and serum markers for e.g. protein glycation and oxidation. Although HF feeding increased body weight, HF and LF mice did not differ in indirect calorimetric values under normoxic conditions and in a fasting state. Exposure to OxR; however, increased oxygen consumption and lipid oxidation in HF mice versus LF mice. Furthermore, OxR induced gluconeogenesis and an antioxidant response in the liver of HF mice, whereas it induced de novo lipogenesis and an antioxidant response in eWAT of LF mice, indicating that HF and LF mice differed in their adaptation to OxR. OxR also increased serum markers of protein glycation and oxidation in HF mice, whereas these changes were absent in LF mice. Cumulatively, OxR is a promising new method to test food products on potential beneficial effects for human health

Decreased methylglyoxal-mediated protein glycation in the healthy aging mouse model of ectopic expression of UCP1 in skeletal muscle

Mice with ectopic expression of uncoupling protein-1 (UCP1) in skeletal muscle exhibit a healthy aging phenotype with increased longevity and resistance to impaired metabolic health. This may be achieved by decreasing protein glycation by the reactive metabolite, methylglyoxal (MG). We investigated protein glycation and oxidative damage in skeletal muscle of mice with UCP1 expression under control of the human skeletal actin promoter (HSA-mUCP1) at age 12 weeks (young) and 70 weeks (aged). We found both young and aged HSA-mUCP1 mice had decreased advanced glycation endproducts (AGEs) formed from MG, lysine-derived N (1-carboxyethyl)lysine (CEL) and arginine-derived hydroimidazolone, MG-H1, whereas protein glycation by glucose forming N -fructosyl-lysine (FL) was increased ca. 2-fold, compared to wildtype controls. There were related increases in FL-linked AGEs, N -carboxymethyl-lysine (CML) and 3-deoxylglucosone-derived hydroimidazolone 3DG-H, and minor changes in protein oxidative and nitration adducts. In aged HSA-mUCP1 mice, urinary MG-derived AGEs/FL ratio was decreased ca. 60% whereas there was no change in CML/FL ratio - a marker of oxidative damage. This suggests that, normalized for glycemic status, aged HSA-mUCP1 mice had a lower flux of whole body MG-derived AGE exposure compared to wildtype controls. Proteomics analysis of skeletal muscle revealed a shift to increased heat shock proteins and mechanoprotection and repair in HSA-mUCP1 mice. Decreased MG-derived AGE protein content in skeletal muscle of aged HSA-mUCP1 mice is therefore likely produced by increased proteolysis of MG-modified proteins and increased proteostasis surveillance of the skeletal muscle proteome. From this and previous transcriptomic studies, signaling involved in enhanced removal of MG-modified protein is likely increased HSPB1-directed HUWE1 ubiquitination through eIF2α-mediated, ATF5-induced increased expression of HSPB1. Decreased whole body exposure to MG-derived AGEs may be linked to increased weight specific physical activity of HSA-mUCP1 mice. Decreased formation and increased clearance of MG-derived AGEs may be associated with healthy aging in the HSA-mUCP1 mouse

Glycation marker glucosepane increases with the progression of osteoarthritis and correlates with morphological and functional changes of cartilage in vivo

Background: Changes of serum concentrations of glycated, oxidized, and nitrated amino acids and hydroxyproline and anticyclic citrullinated peptide antibody status combined by machine learning techniques in algorithms have recently been found to provide improved diagnosis and typing of early-stage arthritis of the knee, including osteoarthritis (OA), in patients. The association of glycated, oxidized, and nitrated amino acids released from the joint with development and progression of knee OA is unknown. We studied this in an OA animal model as well as interleukin-1β-activated human chondrocytes in vitro and translated key findings to patients with OA. Methods: Sixty male 3-week-old Dunkin-Hartley guinea pigs were studied. Separate groups of 12 animals were killed at age 4, 12, 20, 28 and 36 weeks, and histological severity of knee OA was evaluated, and cartilage rheological properties were assessed. Human chondrocytes cultured in multilayers were treated for 10 days with interleukin-1β. Human patients with early and advanced OA and healthy controls were recruited, blood samples were collected, and serum or plasma was prepared. Serum, plasma, and culture medium were analyzed for glycated, oxidized, and nitrated amino acids. Results: Severity of OA increased progressively in guinea pigs with age. Glycated, oxidized, and nitrated amino acids were increased markedly at week 36, with glucosepane and dityrosine increasing progressively from weeks 20 and 28, respectively. Glucosepane correlated positively with OA histological severity (r = 0.58, p < 0.0001) and instantaneous modulus (r = 0.52–0.56; p < 0.0001), oxidation free adducts correlated positively with OA severity (p < 0.0009–0.0062), and hydroxyproline correlated positively with cartilage thickness (p < 0.0003–0.003). Interleukin-1β increased the release of glycated and nitrated amino acids from chondrocytes in vitro. In clinical translation, plasma glucosepane was increased 38% in early-stage OA (p < 0.05) and sixfold in patients with advanced OA (p < 0.001) compared with healthy controls. Conclusions: These studies further advance the prospective role of glycated, oxidized, and nitrated amino acids as serum biomarkers in diagnostic algorithms for early-stage detection of OA and other arthritic disease. Plasma glucosepane, reported here for the first time to our knowledge, may improve early-stage diagnosis and progression of clinical OA

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE−/−) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted. Keywords: Non-alcoholic fatty liver disease, Glyoxalase, Methylglyoxal, Proteomics, iTRA

Experimental characterisation of textile compaction response: A benchmark exercise

This paper reports the results of an international benchmark exercise on the measurement of fibre bed compaction behaviour. The aim was to identify aspects of the test method critical to obtain reliable results and to arrive at a recommended test procedure for fibre bed compaction measurements. A glass fibre 2/2 twill weave and a biaxial (±45°) glass fibre non-crimp fabric (NCF) were tested in dry and wet conditions. All participants used the same testing procedure but were allowed to use the testing frame, the fixture and sample geometry of their choice. The results showed a large scatter in the maximum compaction stress between participants at the given target thickness, with coefficients of variation ranging from 38% to 58%. Statistical analysis of data indicated that wetting of the specimen significantly affected the scatter in results for the woven fabric, but not for the NCF. This is related to the fibre mobility in the architectures in both fabrics. As isolating the effect of other test parameters on the results was not possible, no statistically significant effect of other test parameters could be proven. The high sensitivity of the recorded compaction pressure near the minimum specimen thickness to changes in specimen thickness suggests that small uncertainties in thickness can result in large variations in the maximum value of the compaction stress. Hence, it is suspected that the thickness measurement technique used may have an effect on the scatter

The science case and challenges of space-borne sub-millimeter interferometry

Interstellar matter and star formatio