Johan H. Faber

Human metabolic profiles are stably controlled by genetic and environmental variation.

1H Nuclear Magnetic Resonance spectroscopy (1H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top‐down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non‐identical twin pairs donated plasma and urine samples longitudinally. We acquired 1H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common‐environmental), individual‐environmental, and longitudinally unstable components. We estimate that stable variation, comprising familial and individual‐environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in 1H NMR‐detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker‐discovery studies. We provide a power‐calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect 1H NMR‐based biomarkers quantifying predisposition to disease.

Factor VIII C1-domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake

Background: Antibody KM33 blocks factor VIII (FVIII) endocytosis and phospholipid binding. Results: Hydrogen-deuterium exchange mass spectrometry reveals that KM33 binds C1 domain spikes 2092–2093 and 2158–2159. Glycosylated FVIII-R2159N shows reduced endocytosis and decreased binding to phospholipid membranes with low phosphatidylserine content. Conclusion: Spikes 2092–2093 and 2158–2159 modulate FVIII endocytosis and phospholipid binding. Significance: Novel insight is obtained about the role of the C1 domain for FVIII biology. The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent “hot spot” on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091–2104 and 2157–2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092–2093 and 2158–2159. Spike 2092–2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158–2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092–2093 and 2158–2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology.

Metabonomics in Diabetes Research

Metabonomics has been defined as “quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification” and can provide information on disease processes, drug toxicity, and gene function. In this approach many samples of biological origin (biofluids such as urine or plasma) are analyzed using techniques that produce simultaneous detection. A variety of analytical metabolic profiling tools are used routinely, are also currently under development, and include proton nuclear magnetic resonance spectroscopy and mass spectrometry with a prior online separation step such as high-performance liquid chromatography, ultra-performance liquid chromatography, or gas chromatography. Data generated by these analytical techniques are often combined with multivariate data analysis, i.e., pattern recognition, for respectively generating and interpreting the metabolic profiles of the investigated samples. Metabonomics has gained great prominence in diabetes research within the last few years and has already been applied to understand the metabolism in a range of animal models and, more recently, attempts have been done to process complex metabolic data sets from clinical studies. A future hope for the metabonomic approach is the identification of biomarkers that are able to highlight individuals likely to suffer from diabetes and enable early diagnosis of the disease or the identification of those at risk. This review summarizes the technologies currently being used in metabonomics, as well as the studies reported related to diabetes prior to a description of the general objective of the research plan of the metabonomics part of the European Union project, Molecular Phenotyping to Accelerate Genomic Epidemiology.

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

Background: It is unclear how the LDL receptor family binds large protein ligands. Results: HDX and lysine scanning identified factor (F)VIII regions and specific lysine residues binding low-density lipoprotein receptor-related protein 1 (LRP1). Conclusion: FVIII-LRP1 interaction involves multiple “hot-spot” lysine residues in the A3C1 domains. Significance: Our study sheds light on interactions of complex ligands with the LDL receptor family. Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule.

Metabolic Profiling in Maturity-Onset Diabetes of the Young (MODY) and Young Onset Type 2 Diabetes Fails to Detect Robust Urinary Biomarkers

It is important to identify patients with Maturity-onset diabetes of the young (MODY) as a molecular diagnosis determines both treatment and prognosis. Genetic testing is currently expensive and many patients are therefore not assessed and are misclassified as having either type 1 or type 2 diabetes. Biomarkers could facilitate the prioritisation of patients for genetic testing. We hypothesised that patients with different underlying genetic aetiologies for their diabetes could have distinct metabolic profiles which may uncover novel biomarkers. The aim of this study was to perform metabolic profiling in urine from patients with MODY due to mutations in the genes encoding glucokinase (GCK) or hepatocyte nuclear factor 1 alpha (HNF1A), type 2 diabetes (T2D) and normoglycaemic control subjects. Urinary metabolic profiling by Nuclear Magnetic Resonance (NMR) and ultra performance liquid chromatography hyphenated to Q-TOF mass spectrometry (UPLC-MS) was performed in a Discovery set of subjects with HNF1A-MODY (n = 14), GCK-MODY (n = 17), T2D (n = 14) and normoglycaemic controls (n = 34). Data were used to build a valid partial least squares discriminate analysis (PLS-DA) model where HNF1A-MODY subjects could be separated from the other diabetes subtypes. No single metabolite contributed significantly to the separation of the patient groups. However, betaine, valine, glycine and glucose were elevated in the urine of HNF1A-MODY subjects compared to the other subgroups. Direct measurements of urinary amino acids and betaine in an extended dataset did not support differences between patients groups. Elevated urinary glucose in HNF1A-MODY is consistent with the previously reported low renal threshold for glucose in this genetic subtype. In conclusion, we report the first metabolic profiling study in monogenic diabetes and show that, despite the distinct biochemical pathways affected, there are unlikely to be robust urinary biomarkers which distinguish monogenic subtypes from T2D. Our results have implications for studies investigating metabolic profiles in complex traits including T2D.

Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen–Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.

Membrane Interaction of the Factor VIIIa Discoidin Domains in Atomistic Detail.

A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor VIIIa (FVIIIa), the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly was positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla) domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent nonspecificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2.