Jag har plöjt igenom denna snåriga studie ett antal gånger för att försöka få ett grepp om dess innehåll och hur den berör GlycoImaging.Man behöver nästan vara forskare själv för att förstå helheten och själva essensen av vad forskarna kommit fram till. 😎
Abstract
Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.
Introduction
Nature’s overarching solution for fulfilling the need for symbiosis with a vast community of microorganisms—our microbiomes—are mucins1,2,3. Mucins in the gut constitute the primary barrier as well as the ecological niche for the microbiome. Dynamic replenishment of mucin layers provides a constant selection of the resident microbiome through adhesive interactions, and degradation of mucin O-glycans by members of the microbiota supply nutrients. Mucins are a large family of heavily glycosylated proteins that line all mucosal surfaces and represent the major macromolecules in body fluids2. Mucins clear, contain, feed, direct, and continuously replenish our microbiomes, limiting unwanted co-habitation and repressing harmful pathogenic microorganisms. Recent studies show that specific mucins can disperse biofilms and disrupt bacterial aggregation, but the molecular basis of these effects remains largely obscure. Mucin O-glycans present the essential binding opportunities and informational cues for microorganisms via adhesins, however, our understanding of these features is essentially limited to results from studies with simple oligosaccharides without the protein context of mucins and the higher-order features presented by dense O-glycan motifs. Mucins are notoriously difficult to isolate due to their size and heterogeneity, and production by recombinant expression in cell lines is impeded due to difficulties with the assembly of full coding expression plasmids often resulting in heterogeneous products.
State-of-the-art technologies to capture the informational content of mucins are confined to studies with synthetic and isolated O-glycans, synthetic and chemoenzymatically produced short glycopeptides, and synthetic glycopeptides as well as non-natural polymers; all of which are rare commodities that do not reflect the complex information captured in distinct human mucins by their display of patterns and structures of O-glycans. With the advent of the facile nuclease-based gene engineering technologies, it has become possible to engineer mammalian cells with combinatorial knockout (KO) and knockin (KI) of glycosylation genes to display subsets and distinct features of the glycome on the cell surface or on secreted reporter proteins in order to probe biological interactions dependent on glycans. Genetic engineering provides opportunities for interpretation and dissection of the glycosylation genes, biosynthetic pathways, and structural features required for identifiable interactions with the cell library17. Importantly, such cell-based display strategies allow for the presentation of glycans in the natural context of glycoproteins and the cell surface, and this has provided the first experimental evidence for the existence of higher-order binding motifs consisting of O-glycans in dense patterns.
There are at least 18 distinct mucin genes encoding membrane or secreted mucins. The large gel-forming secreted mucins may form oligomeric networks or extended bundles through inter- and intramolecular disulfide bridges in the C- and N-terminal cysteine-rich regions. A common characteristic of all mucins is that the major part of their extracellular region comprises a variable number of imperfect tandem repeated (TR) sequences (also called PTS sequences) that carry dense O-glycans, with the notable exception of MUC16 that contains a large, densely O-glycosylated N-terminal region without TRs. Arguably, the main cues for the microbiota lie in the TR regions that display O-glycans of diverse structures and positions in unique patterns. Interestingly, the TR regions appear poorly conserved throughout evolution in contrast to the flanking regions of the large mucins, which has been interpreted to reflect that the TR regions simply need to carry dense O-glycans without specific patterns. An alternative interpretation is that the divergence in TR sequences has co-evolved with the microbiota to govern refined interactions with larger motifs of O-glycan patterns as recently suggested for streptococcal serine-rich adhesins. The TR regions of mucins are quite distinct in length and in sequences with a distinct spacing of O-glycosites, and TRs in any mucin exhibit individual variability in numbers as well as to some degree in actual sequences21. Thus, there are rich opportunities for unique codes in mucin TRs, governed by the particular display of patterns and structures of O-glycans. The mucin TRs and their glycocodes may be considered the informational content of mucins and thus comprise the mucinome. The TR mucinome provides a much greater potential binding epitome than the comparatively limited repertoire of binding epitopes comprised of simple oligosaccharide motifs available in humans.
We developed a cell-based platform for the display and production of representative mucin TRs with defined O-glycans.
Discussion
The cell-based mucin display platform presented here offers a unique resource with wide applications and opportunities for the discovery and dissection of molecular properties of natural human mucins and other glycoproteins with mucin domains. The informational cues harbored in mucin TRs with their distinct patterns and structures of O-glycans can now be addressed with well-defined molecules in a variety of assay formats. This was illustrated by analyzing the binding properties of streptococcal Siglec-like adhesins revealing requirements for specific O-glycan structures and select mucin TR sequences. We used the mucin display to dissect the fine substrate specificity of the mucin-destroying glycoprotease StcE derived from pathogenic EHEC, demonstrating clear selectivity for both distinct mucin TRs and O-glycoforms, and importantly discovering that the normal core3 O-glycosylation pathway in the colon actually inhibits StcE digestion of MUC2 . Studies with IAV also suggest that binding to and release from mucins are not simply determined by the number of O-glycans and sialic acids, but partly driven by cues encoded by the TR sequences and O-glycan structure . The mucin TR reporter production platform also provided unprecedented insights into the O-glycosylation of mucins and enabled intact MS analysis to demonstrate surprising efficiency in the initiation of O-glycosylation by the polypeptide GalNAc-Ts."
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