Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase.

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

ALPK1 mutants causing ROSAH syndrome or Spiradenoma are activated by human nucleotide sugars.

The atypical protein kinase ALPK1 is activated by the bacterial nucleotide sugar ADP-heptose and phosphorylates TIFA to switch on a signaling pathway that combats microbial infection. In contrast, ALPK1 mutations cause two human diseases: the ALPK1[T237M] and ALPK1[Y254C] mutations underlie ROSAH syndrome (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis, and migraine headache), while the ALPK1[V1092A] mutation accounts for 45% of spiradenoma and 30% of spiradenocarcinoma cases studied. In this study, we demonstrate that unlike wild-type (WT) ALPK1, the disease-causing ALPK1 mutants trigger the TIFA-dependent activation of an NF-κB/activator protein 1 reporter gene in the absence of ADP-heptose, which can be suppressed by either of two additional mutations in the ADP-heptose binding site that prevent the activation of WT ALPK1 by ADP-heptose. These observations are explained by our key finding that although ALPK1[T237M] and ALPK1[V1092A] are activated by bacterial ADP-heptose, they can also be activated by nucleotide sugars present in human cells (UDP-mannose, ADP-ribose, and cyclic ADP-ribose) which can be prevented by disruption of the ADP-heptose binding site. The ALPK1[V1092A] mutant was also activated by GDP-mannose, which did not activate ALPK1[T237M]. These are new examples of disease-causing mutations permitting the allosteric activation of an enzyme by endogenous molecules that the WT enzyme does not respond to. We propose that the loss of the specificity of ALPK1 for bacterial ADP-heptose underlies ROSAH syndrome and spiradenoma/spiradenocarcinoma caused by ALPK1 mutation.

The D-mannose/L-galactose pathway plays a predominant role in ascorbate biosynthesis in the liverwort Marchantia polymorpha but is not regulated by light and oxidative stress.

Ascorbate plays an indispensable role in plants, functioning as both an antioxidant and a cellular redox buffer. It is widely acknowledged that the ascorbate biosynthesis in the photosynthetic tissues of land plants is governed by light-mediated regulation of the D-mannose/L-galactose (D-Man/L-Gal) pathway. At the core of this light-dependent regulation lies the VTC2 gene, encoding the rate-limiting enzyme GDP-L-Gal phosphorylase. The VTC2 expression is regulated by signals via the photosynthetic electron transport system. In this study, we directed our attention to the liverwort Marchantia polymorpha, representing one of the basal land plants, enabling us to conduct an in-depth analysis of its ascorbate biosynthesis. The M. polymorpha genome harbors a solitary gene for each enzyme involved in the D-Man/L-Gal pathway, including VTC2, along with three lactonase orthologs, which may be involved in the alternative ascorbate biosynthesis pathway. Through supplementation experiments with potential precursors, we observed that only L-Gal exhibited effectiveness in ascorbate biosynthesis. Furthermore, the generation of VTC2-deficient mutants through genome editing unveiled the inability of thallus regeneration in the absence of L-Gal supplementation, thereby revealing the importance of the D-Man/L-Gal pathway in ascorbate biosynthesis within M.  polymorpha. Interestingly, gene expression analyses unveiled a distinct characteristic of M. polymorpha, where none of the genes associated with the D-Man/L-Gal pathway, including VTC2, showed upregulation in response to light, unlike other known land plants. This study sheds light on the exceptional nature of M. polymorpha as a land plant that has evolved distinctive mechanisms concerning ascorbate biosynthesis and its regulation.

Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Role of succinyl substituents in the mannose-capping of lipoarabinomannan and control of inflammation in Mycobacterium tuberculosis infection.

The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.

Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein.

Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.

High-Mannose-Type Glycan of Basigin in Endothelial Cells Is Essential for the Opening of the Blood-Brain Barrier Induced by Hypoxia, Cyclophilin A, or Tumor Necrosis Factor-α.

Pathologic opening of the blood-brain barrier accelerates the progression of various neural diseases. Basigin, as an essential molecule for the opening of the blood-brain barrier, is a highly glycosylated transmembrane molecule specified in barrier-forming endothelial cells. This study analyzed the involvement of basigin in the regulation of the blood-brain barrier focusing on its glycosylation forms. First, basigin was found to be expressed as cell surface molecules with complex-type glycan as well as those with high-mannose-type glycan in barrier-forming endothelial cells. Monolayers of endothelial cells with suppressed expression of basigin with high-mannose-type glycan were then prepared and exposed to pathologic stimuli. These monolayers retained their barrier-forming properties even in the presence of pathologic stimuli, although their expression of basigin with complex-type glycan was maintained. In vivo, the blood-brain barrier in mice pretreated intravenously with endoglycosidase H was protected from opening under pathologic stimuli. Pathologically opened blood-brain barrier in streptozotocin-injected mice was successfully closed by intravenous injection of endoglycosidase H. These results show that high-mannose-type glycan of the basigin molecule is essential for the opening of the blood-brain barrier and therefore a specific target for protection as well as restoration of pathologic opening of the blood-brain barrier.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles.

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV.

N-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N-glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N-glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.

The PilT retraction ATPase promotes both extension and retraction of the MSHA type IVa pilus in Vibrio cholerae.

Diverse bacterial species use type IVa pili (T4aP) to interact with their environments. The dynamic extension and retraction of T4aP is critical for their function, but the mechanisms that regulate this dynamic activity remain poorly understood. T4aP are typically extended via the activity of a dedicated extension motor ATPase and retracted via the action of an antagonistic retraction motor ATPase called PilT. These motors are generally functionally independent, and loss of PilT commonly results in T4aP hyperpiliation due to undeterred pilus extension. However, for the mannose-sensitive hemagglutinin (MSHA) T4aP of Vibrio cholerae, the loss of PilT unexpectedly results in a loss of surface piliation. Here, we employ a combination of genetic and cell biological approaches to dissect the underlying mechanism. Our results demonstrate that PilT is necessary for MSHA pilus extension in addition to its well-established role in promoting MSHA pilus retraction. Through a suppressor screen, we also provide genetic evidence that the MshA major pilin impacts pilus extension. Together, these findings contribute to our understanding of the factors that regulate pilus extension and describe a previously uncharacterized function for the PilT motor ATPase.

D-mannose facilitates immunotherapy and radiotherapy of triple-negative breast cancer via degradation of PD-L1.

Breast cancer is the most frequent malignancy in women worldwide, and triple-negative breast cancer (TNBC) patients have the worst prognosis and highest risk of recurrence. The therapeutic strategies for TNBC are limited. It is urgent to develop new methods to enhance the efficacy of TNBC treatment. Previous studies demonstrated that D-mannose, a hexose, can enhance chemotherapy in cancer and suppress the immunopathology of autoimmune diseases. Here, we show that D-mannose can significantly facilitate TNBC treatment via degradation of PD-L1. Specifically, D-mannose can activate AMP-activated protein kinase (AMPK) to phosphorylate PD-L1 at S195, which leads to abnormal glycosylation and proteasomal degradation of PD-L1. D-mannose-mediated PD-L1 degradation promotes T cell activation and T cell killing of tumor cells. The combination of D-mannose and PD-1 blockade therapy dramatically inhibits TNBC growth and extends the lifespan of tumor-bearing mice. Moreover, D-mannose-induced PD-L1 degradation also results in messenger RNA destabilization of DNA damage repair-related genes, thereby sensitizing breast cancer cells to ionizing radiation (IR) treatment and facilitating radiotherapy of TNBC in mice. Of note, the effective level of D-mannose can be easily achieved by oral administration in mice. Our study unveils a mechanism by which D-mannose targets PD-L1 for degradation and provides methods to facilitate immunotherapy and radiotherapy in TNBC. This function of D-mannose may be useful for clinical treatment of TNBC.

Antioxidant and Immunomodulatory Polymer Vesicles for Effective Diabetic Wound Treatment through ROS Scavenging and Immune Modulating.

Chronic diabetic wound patients usually show high glucose levels and systemic immune disorder, resulting in high reactive oxygen species (ROS) levels and immune cell dysfunction, prolonged inflammation, and delayed wound healing. Herein, we prepared an antioxidant and immunomodulatory polymer vesicle for diabetic wound treatment. This vesicle is self-assembled from poly(ε-caprolactone)36-block-poly[lysine4-stat-(lysine-mannose)22-stat-tyrosine)16] ([PCL36-b-P[Lys4-stat-(Lys-Man)22-stat-Tyr16]). Polytyrosine is an antioxidant polypeptide that can scavenge ROS. d-Mannose was introduced to afford immunomodulatory functions by promoting macrophage transformation and Treg cell activation through inhibitory cytokines. The mice treated with polymer vesicles showed 23.7% higher Treg cell levels and a 91.3% higher M2/M1 ratio than those treated with PBS. Animal tests confirmed this vesicle accelerated healing and achieved complete healing of S. aureus-infected diabetic wounds within 8 days. Overall, this is the first antioxidant and immunomodulatory vesicle for diabetic wound healing by scavenging ROS and regulating immune homeostasis, opening new avenues for effective diabetic wound healing.

Chromatograms and Mass Spectra of High-Mannose and Paucimannose N-Glycans for Rapid Isomeric Identifications.

N-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, N-glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MSn mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose N-glycan isomers. These N-glycans include isomers by breaking of arbitrary numbers of glycosidic bonds at arbitrary positions of canonical Man9GlcNAc2 N-glycans. In addition, some GlcMannGlcNAc2 N-glycan isomers were included in the database. This database is particularly useful for the identification of the N-glycans not in conventional N-glycan standards. This study demonstrated the application of the database to structural assignment for high-mannose N-glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma cells. We found many N-glycans that are not expected to be generated by conventional biosynthetic pathways of multicellular eukaryotes.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations.

The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called FimH FocH , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and FimH FocH are almost identical, experimental evidence shows that FimH FocH is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in FimH FocH . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in FimH FocH than FimH. In conclusion, the mutations in FimH FocH shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.

Spatial N-glycomics of the normal breast microenvironment reveals fucosylated and high-mannose N-glycan signatures related to BI-RADS density and ancestry.

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.