Complex metabolic disharmony in PMM2-CDG paves the way to new therapeutic approaches.

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients.

Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry.

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

Efficient generation of peptide hydrazides via direct hydrazinolysis of Peptidyl-Wang-TentaGel resins.

Peptide hydrazides are valuable building blocks in peptide and protein chemistry, e.g. as precursors of peptide thioesters that allow the preparation of these important intermediates under mild conditions. Additional robust and versatile methods for the generation of peptide hydrazides from standard solid supports are therefore highly desired in order to facilitate access to peptide thioester via Fmoc-based SPPS. Here, the efficient generation of peptide hydrazides from conventional 4-hydroxymethyl phenol Wang-TentalGel peptidyl resins is described. Direct hydrazinolysis of a 19mer mucin1 peptide gives the protected peptide hydrazide in excellent yields. Testing a series of octapeptides carrying the 20 common proteinogenic amino acids at their C-terminus led to preparation of all corresponding peptide hydrazides in very good to excellent yields and purities. The available set of octapeptides allowed analyzing the influence of the nature of the C-terminal amino acid and of the solvent on the hydrazinolysis reaction. Furthermore, the compatibility of the method with posttranslational modifications (here glycosylation) and with potentially sensitive functional groups in amino acid side chains makes this approach a viable alternative for obtaining peptide hydrazides. It combines the advantages of a straightforward synthesis with stereochemical stability and flexibility, as it provides easy access to the peptide acid and the peptide thioester (via the hydrazide) from the same solid support.