Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>107 mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency (kcat/Km) for the synthesis of Lewis x and 3′-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.

In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins.

Hair follicle dermal sheath (DS) harbors hair follicle dermal stem cells (hfDSCs), which can be recruited to replenish DS and dermal papilla (DP). Cultured DS cells can differentiate into various cell lineages in vitro. However, it is unclear how its plasticity is modulated in vivo. Wnt/β-catenin signaling plays an important role in maintaining stem cells of various lineages and is required for HF development and regeneration. Here we report that activation of β-catenin in DS generates ectopic HF outgrowth (EF) by reprogramming HF epidermal cells and DS cells themselves, and endows DS cells with hair inducing ability. Epidermal homeostasis of pre-existing HFs is disrupted. Additionally, cell-autonomous progressive skin fibrosis is prominent in dermis, where the excessive fibroblasts largely originate from DS. Gene expression analysis of purified DS cells with activated β-catenin revealed significantly increased expression of Bmp, Fgf, and Notch ligands and administration of Bmp, Fgf, or Notch signaling inhibitor attenuates EF formation. In summary, our findings advance the current knowledge of high plasticity of DS cells and provide an insight into understanding how Wnt/β-catenin signaling controls DS cell behaviors.

No abstract

Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn(2+) and Cu(2+), while Fe(2+), Co(2+), Ni(2+), Mn(2+), Ca(2+), and Mg(2+) promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids.

The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (codes 5CCU, 5J7Z, and 5J14) have been deposited in the Protein Data Bank (http://wwpdb.org/). □ S This article contains supplemental Tables S1-S3 and Figs. S1-S4.

No abstract

Consecutive glycosylation sites occur in both self and viral proteins. Glycan-shielding of underneath peptide region is a double-edged sword, that avoids immune attack to self-proteins, but helps viruses including HIV-1 and SARS-CoV2 to escape antibody binding. Here we report a high-affinity antibody, 16A, binding to linear peptide containing consecutive glycosylation sites. Co-crystallization of 16A Fab and glycopeptides with GalNAc modifications at different sites showed that STAPPAHG is the sequence recognized by 16A antibody. GalNAc modification at Threonine site on STAPPAHG sequence significantly increased the affinity of Fab binding by 30.6 fold (KD = 6.7nM). The increased affinity is conferred by hydrophilic and pi-stacking interactions between the GalNAc residue on Threonine site and a Trp residue from the CDR1 region of the heavy chain. Furthermore, molecular modeling suggested that GalNAc on T site causes more favorable conformation for antibody binding. These results showed that glycan modification most proximal to linear peptide core epitope significantly increases antigenicity of a glycopeptide epitope. The antibody recognition mode by peptide-binding CDR groove with a glycan-binding edge, may shed light on designing of linear glycopeptide-based vaccines for cancer and viral diseases.