Non-targeted isomer-sensitive N-glycome analysis reveals new layers of organ-specific diversity in mice.

N-glycosylation is one of the most common protein modifications in eukaryotes, with immense importance at the molecular, cellular, and organismal level. Accurate and reliable N-glycan analysis is essential to obtain a systems-wide understanding of fundamental biological processes. Due to the structural complexity of glycans, their analysis is still highly challenging. Here we make publicly available a consistent N-glycome dataset of 20 different mouse tissues and demonstrate a multimodal data analysis workflow that allows for unprecedented depth and coverage of N-glycome features. This highly scalable, LC-MS/MS data-driven method integrates the automated identification of N-glycan spectra, the application of non-targeted N-glycome profiling strategies and the isomer-sensitive analysis of glycan structures. Our delineation of critical sub-structural determinants and glycan isomers across the mouse N-glycome uncovered tissue-specific glycosylation patterns, the expression of non-canonical N-glycan structures and highlights multiple layers of N-glycome complexity that derive from organ-specific regulations of glycobiological pathways.

Introduction of a human- and keyboard-friendly N-glycan nomenclature.

In the beginning was the word. But there were no words for N-glycans, at least, no simple words. Next to chemical formulas, the IUPAC code can be regarded as the best, most reliable and yet immediately comprehensible annotation of oligosaccharide structures of any type from any source. When it comes to N-glycans, the venerable IUPAC code has, however, been widely supplanted by highly simplified terms for N-glycans that count the number of antennae or certain components such as galactoses, sialic acids and fucoses and give only limited room for exact structure description. The highly illustrative - and fortunately now standardized - cartoon depictions gained much ground during the last years. By their very nature, cartoons can neither be written nor spoken. The underlying machine codes (e.g., GlycoCT, WURCS) are definitely not intended for direct use in human communication. So, one might feel the need for a simple, yet intelligible and precise system for alphanumeric descriptions of the hundreds and thousands of N-glycan structures. Here, we present a system that describes N-glycans by defining their terminal elements. To minimize redundancy and length of terms, the common elements of N-glycans are taken as granted. The preset reading order facilitates definition of positional isomers. The combination with elements of the condensed IUPAC code allows to describe even rather complex structural elements. Thus, this "proglycan" coding could be the missing link between drawn structures and software-oriented representations of N-glycan structures. On top, it may greatly facilitate keyboard-based mining for glycan substructures in glycan repositories.

Simple Routes to Stable Isotope-Coded Native Glycans.

Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13C-containing N-acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC-MS on either porous graphitic carbon or─following permethylation─on reversed phase.

Towards Mapping of the Human Brain N-Glycome with Standardized Graphitic Carbon Chromatography.

The brain N-glycome is known to be crucial for many biological functions, including its involvement in neuronal diseases. Although large structural studies of brain N-glycans were recently carried out, a comprehensive isomer-specific structural analysis has still not been achieved, as indicated by the recent discovery of novel structures with galactosylated bisecting GlcNAc. Here, we present a detailed, isomer-specific analysis of the human brain N-glycome based on standardized porous graphitic carbon (PGC)-LC-MS/MS. To achieve this goal, we biosynthesized glycans with substitutions typically occurring in the brain N-glycome and acquired their normalized retention times. Comparison of these values with the standardized retention times of neutral and desialylated N-glycan fractions of the human brain led to unambiguous isomer specific assignment of most major peaks. Profound differences in the glycan structures between naturally neutral and desialylated glycans were found. The neutral and sialylated N-glycans derive from diverging biosynthetic pathways and are biosynthetically finished end products, rather than just partially processed intermediates. The focus on structural glycomics defined the structure of human brain N-glycans, amongst these are HNK-1 containing glycans, a bisecting sialyl-lactose and structures with fucose and N-acetylgalactosamine on the same arm, the so-called LDNF epitope often associated with parasitic worms.

Substrate specificity and complex stability of coproporphyrin ferrochelatase is governed by hydrogen-bonding interactions of the four propionate groups.

Coproporpyhrin III is the substrate of coproporphyrin ferrochelatases (CpfCs). These enzymes catalyse the insertion of ferrous iron into the porphyrin ring. This is the penultimate step within the coproporphyrin-dependent haeme biosynthesis pathway. This pathway was discovered in 2015 and is mainly utilised by monoderm bacteria. Prior to this discovery, monoderm bacteria were believed to utilise the protoporphyrin-dependent pathway, analogously to diderm bacteria, where the substrate for the respective ferrochelatase is protoporphyrin IX, which has two propionate groups at positions 6 and 7 and two vinyl groups at positions 2 and 4. In this work, we describe for the first time the interactions of the four-propionate substrate, coproporphyrin III, and the four-propionate product, iron coproporphyrin III (coproheme), with the CpfC from Listeria monocytogenes and pin down differences with respect to the protoporphyrin IX and haeme b complexes in the wild-type (WT) enzyme. We further created seven LmCpfC variants aiming at altering substrate and product coordination. The WT enzyme and all the variants were comparatively studied by spectroscopic, thermodynamic and kinetic means to investigate in detail the H-bonding interactions, which govern complex stability and substrate specificity. We identified a tyrosine residue (Y124 in LmCpfC), coordinating the propionate at position 2, which is conserved in monoderm CpfCs, to be highly important for binding and stabilisation. Importantly, we also describe a tyrosine-serine-threonine triad, which coordinates the propionate at position 4. The study of the triad variants indicates structural differences between the coproporphyrin III and the coproheme complexes. ENZYME: EC 4.99.1.9.

Bisecting Lewis X in Hybrid-Type N-Glycans of Human Brain Revealed by Deep Structural Glycomics.

The importance of protein glycosylation in the biomedical field requires methods that not only quantitate structures by their monosaccharide composition, but also resolve and identify the many isomers expressed by mammalian cells. The art of unambiguous identification of isomeric structures in complex mixtures, however, did not yet catch up with the fast pace of advance of high-throughput glycomics. Here, we present a strategy for deducing structures with the help of a deci-minute accurate retention time library for porous graphitic carbon chromatography with mass spectrometric detection. We implemented the concept for the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of the 40 biosynthetized isomers occupied unique elution positions. This result demonstrates the unique isomer selectivity of porous graphitic carbon. With the help of a rather tightly spaced grid of isotope-labeled internal N-glycan, standard retention times were transposed to a standard chromatogram. Application of this approach to animal and human brain N-glycans immediately identified the majority of structures as being of the bisected type. Most notably, it exposed hybrid-type glycans with galactosylated and even Lewis X containing bisected N-acetylglucosamine, which have not yet been discovered in a natural source. Thus, the time grid approach implemented herein facilitated discovery of the still missing pieces of the N-glycome in our most noble organ and suggests itself─in conjunction with collision induced dissociation─as a starting point for the overdue development of isomer-specific deep structural glycomics.

Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase.

Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram-positive) bacteria within the recently detected coproporphyrin-dependent (CPD) heme biosynthesis pathway. Here, we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme-bound CpfC. Comparison of crystal structures of apo-LmCpfC and coproheme-LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo-, coproporphyrin III-, and coproheme-LmCpfC underline the pronounced noncovalent interaction of both coproporphyrin and coproheme with the protein (ΔTm  = 11 °C compared to apo-LmCpfC), which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53, and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo-LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III-LmCpfC that immediately leads to formation of ferric coproheme-LmCpfC (kcat /KM  = 4.7 × 105  m-1 ·s-1 ). We compare the crystal structure of coproheme-LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the CPD heme biosynthesis pathway. ENZYME: EC 4.99.1.9 DATABASE: pdb-codes of structural data in this work: 6RWV, 6SV3.