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Heparan sulfate (HS) polysaccharides are master regulators of diverse biological processes via sulfated motifs that can recruit specific proteins. 3-O-sulfation of HS/heparin is crucial for anticoagulant activity, but despite emerging evidence for roles in many other functions, a lack of tools for deciphering structure-function relationships has hampered advances. Here, we describe an approach integrating synthesis of 3-O-sulfated standards, comprehensive HS disaccharide profiling, and cell engineering to address this deficiency. Its application revealed previously unseen differences in 3-O-sulfated profiles of clinical heparins and 3-O-sulfotransferase (HS3ST)–specific variations in cell surface HS profiles. The latter correlated with functional differences in anticoagulant activity and binding to platelet factor 4 (PF4), which underlies heparin-induced thrombocytopenia, a known side effect of heparin. Unexpectedly, cells expressing the HS3ST4 isoenzyme generated HS with potent anticoagulant activity but weak PF4 binding. The data provide new insights into 3-O-sulfate structure-function and demonstrate proof of concept for tailored cell-based synthesis of next-generation heparins.

The degradation of glycosaminoglycans (GAGs) involves a series of exolytic glycosidases and sulfatases that act sequentially on the nonreducing end of the polysaccharide chain. Enzymes have been cloned that catalyze all of the known linkages with the exception of the removal of the 2-O-sulfate group from 2-sulfoglucuronate, which is found in heparan sulfate and dermatan sulfate. Here, we show using synthetic disaccharide substrates that arylsulfatase K is the glucuronate-2-sulfatase. Arylsulfatase K acts selectively on 2-sulfoglucuronate and lacks activity against 2-sulfoiduronate, whereas iduronate-2-sulfatase (IDS) desulfates synthetic disaccharides containing 2-sulfoiduronate but not 2-sulfoglucuronate. As arylsulfatase K has all of the properties expected of a lysosomal enzyme, we conclude that arylsulfatase K is the long sought lysosomal glucuronate-2-sulfatase, which we designate GDS.

Human milk oligosaccharides (HMOs) are complex sugars highly abundant in human milk but currently not present in infant formula. Rapidly accumulating evidence from in vitro and in vivo studies, combined with epidemiological associations and correlations, suggests that HMOs benefit infants through multiple mechanisms and in a variety of clinical contexts. Until recently, however, research on HMOs has been limited by an insufficient availability of HMOs. Most HMOs are found uniquely in human milk, and thus far it has been prohibitively tedious and expensive to isolate and synthesize them. This article reviews new strategies to overcome this lack of availability by generating HMOs through chemoenzymatic synthesis, microbial metabolic engineering, and isolation from human donor milk or dairy streams. Each approach has its advantages and comes with its own challenges, but combining the different methods and acknowledging their limitations creates new opportunities for research and application with the goal of improving maternal and infant health.

Sialyl‐Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty‐two isomeric asymmetrical tri‐antennary N‐glycans presenting SLex‐Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high‐affinity ligands for E‐selectin. The N‐glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5′‐diphospho‐N‐trifluoracetylglucosamine (UDP‐GlcNHTFA) that can be transferred by branching N‐acetylglucosaminyltransferases to give, after base treatment, GlcNH2‐containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E‐selectin bound equally well to linear glycans and tri‐antennary N‐glycans presenting SLex‐Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri‐antennary N‐glycans. Furthermore, several H5 HAs preferentially bound to N‐glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N‐glycans presenting SLex. [ABSTRACT FROM AUTHOR]

Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C‐2 of the lipid A component of LPS induces dimerization of two TLR4‐MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)‐3‐hydroxymyristate at C‐2 of lipid A, a range of bis‐ and mono‐phosphoryl lipid A derivatives with different modifications at C‐2 were prepared by a strategy in which 2‐methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C‐2 amine was protected as 9‐fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C‐2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C‐2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A. [ABSTRACT FROM AUTHOR]

Poly‐N‐acetyl lactosamines (polyLacNAc) are common structural motifs of N‐ and O‐linked glycan, glycosphingolipids and human milk oligosaccharides. They can be branched by the addition of β1,6‐linked N‐acetyl‐glucosamine (GlcNAc) moieties to internal galactoside (Gal) residues by the I‐branching enzyme beta‐1,6‐N‐acetylglucosaminyltransferase 2 (GCNT2). I‐branching has been implicated in many biological processes and is also associated with various diseases such as cancer progression. Currently, there is a lack of methods that can install, in a regioselective manner, I‐branches and allows the preparation of isomeric poly‐LacNAc derivatives. Here, we described a chemo‐enzymatic strategy that addresses this deficiency and is based on the enzymatic assembly of an oligo‐LacNAc chain that at specific positions is modified by a GlcNTFA moiety. Replacement of the trifluoroacetyl (TFA) moiety by tert‐butyloxycarbonyl (Boc) gives compounds in which the galactoside at the proximal site is blocked from modification by GCNT2. After elaboration of the antennae, the Boc group can be removed, and the resulting amine acetylated to give natural I‐branched structures. It is also shown that fucosides can function as a traceless blocking group that can provide complementary I‐branched structures from a single precursor. The methodology made it possible to synthesize a library of polyLacNAc chains having various topologies. [ABSTRACT FROM AUTHOR]