In-Depth Mass Spectrometry Analysis Reveals the Plasma Proteomic and N-Glycoproteomic Impact of an Amish-Enriched Cardioprotective Variant in B4GALT1.

B4GALT1 encodes ß-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal galactose. Studies increasingly suggest that B4GALT1 may be involved in the regulation of lipid metabolism pathways. Recently, we discovered a single-site missense variant Asn352Ser (N352S) in the functional domain of B4GALT1 in an Amish population, which decreases the level of LDL-cholesterol (LDL-c) as well as the protein levels of ApoB, fibrinogen, and IgG in the blood. To systematically evaluate the effects of this missense variant on protein glycosylation, expression, and secretion, we developed a nano-LC-MS/MS-based platform combined with TMT-labeling for in-depth quantitative proteomic and glycoproteomic analyses in the plasma of individuals homozygous for the B4GALT1 missense variant N352S versus non-carriers (n = 5 per genotype). A total of 488 secreted proteins in the plasma were identified and quantified, 34 of which showed significant fold changes in protein levels between N352S homozygotes and non-carriers. We determined N-glycosylation profiles from 370 glycosylation sites in 151 glycoproteins and identified ten proteins most significantly associated with decreased galactosylation and sialyation in B4GALT1 N352S homozygotes. These results further support that B4GALT1 N352S alters the glycosylation profiles of a variety of critical target proteins, thus governing the functions of these proteins in multiple pathways, such as those involved in lipid metabolism, coagulation, and the immune response.

Glycomic analysis of host response reveals high mannose as a key mediator of influenza severity.

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3 to 5 million cases of severe illness and ∼290,000 to 645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannose-dependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity.

miRNA proxy approach reveals hidden functions of glycosylation.

Glycosylation, the most abundant posttranslational modification, holds an unprecedented capacity for altering biological function. Our ability to harness glycosylation as a means to control biological systems is hampered by our inability to pinpoint the specific glycans and corresponding biosynthetic enzymes underlying a biological process. Herein we identify glycosylation enzymes acting as regulatory elements within a pathway using microRNA (miRNA) as a proxy. Leveraging the target network of the miRNA-200 family (miR-200f), regulators of epithelial-to-mesenchymal transition (EMT), we pinpoint genes encoding multiple promesenchymal glycosylation enzymes (glycogenes). We focus on three enzymes, beta-1,3-glucosyltransferase (B3GLCT), beta-galactoside alpha-2,3-sialyltransferase 5 (ST3GAL5), and (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), encoding glycans that are difficult to analyze by traditional methods. Silencing these glycogenes phenocopied the effect of miR-200f, inducing mesenchymal-to-epithelial transition. In addition, all three are up-regulated in TGF-ß-induced EMT, suggesting tight integration within the EMT-signaling network. Our work indicates that miRNA can act as a relatively simple proxy to decrypt which glycogenes, including those encoding difficult-to-analyze structures (e.g., proteoglycans, glycolipids), are functionally important in a biological pathway, setting the stage for the rapid identification of glycosylation enzymes driving disease states.

Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode.

Cell surface glycans form a critical interface with the biological milieu, informing diverse processes from the inflammatory cascade to cellular migration. Assembly of discrete carbohydrate structures requires the coordinated activity of a repertoire of proteins, including glycosyltransferases and glycosidases. Little is known about the regulatory networks controlling this complex biosynthetic process. Recent work points to a role for microRNA (miRNA) in the regulation of specific glycan biosynthetic enzymes. Herein we take a unique systems-based approach to identify connections between miRNA and the glycome. By using our glycomic analysis platform, lectin microarrays, we identify glycosylation signatures in the NCI-60 cell panel that point to the glycome as a direct output of genomic information flow. Integrating our glycomic dataset with miRNA data, we map miRNA regulators onto genes in glycan biosynthetic pathways (glycogenes) that generate the observed glycan structures. We validate three of these predicted miRNA/glycogene regulatory networks: high mannose, fucose, and terminal ß-GalNAc, identifying miRNA regulation that would not have been observed by traditional bioinformatic methods. Overall, our work reveals critical nodes in the global glycosylation network accessible to miRNA regulation, providing a bridge between miRNA-mediated control of cell phenotype and the glycome.

Insights into miRNA regulation of the human glycome.

Glycosylation is an intricate process requiring the coordinated action of multiple proteins, including glycosyltransferases, glycosidases, sugar nucleotide transporters and trafficking proteins. Work by several groups points to a role for microRNA (miRNA) in controlling the levels of specific glycosyltransferases involved in cancer, neural migration and osteoblast formation. Recent work in our laboratory suggests that miRNA are a principal regulator of the glycome, translating genomic information into the glycocode through tuning of enzyme levels. Herein we overlay predicted miRNA regulation of glycosylation related genes (glycogenes) onto maps of the common N-linked and O-linked glycan biosynthetic pathways to identify key regulatory nodes of the glycome. Our analysis provides insights into glycan regulation and suggests that at the regulatory level, glycogenes are non-redundant.

Vaginal Product Formulation Alters the Innate Antiviral Activity and Glycome of Cervicovaginal Fluids with Implications for Viral Susceptibility.

Glycosylated proteins (i.e., mucins, IgG) are important mediators of innate antiviral immunity in the vagina; however, our current knowledge of the role that glycan themselves play in genital immunity is relatively low. Herein, we evaluate the relationship between innate antiviral immunity and glycomic composition in cervicovaginal lavage fluid (CVL) collected as part of a Phase I clinical trial testing the impact of two distinct formulations of the antiretroviral drug dapivirine. Using lectin microarray technology, we discovered that formulation (hydrogel- versus film-based delivery) impacted the CVL glycome, with hydrogel formulations inducing more changes, including a loss of high-mannose. The loss of this epitope correlated to a loss of anti-HIV-1 activity. Glycoproteomic identification of high-mannose proteins revealed a cohort of antiproteases shown to be important in HIV-1 resistance, whose expression covaried with the high-mannose signature. Our data strongly suggests high-mannose as a marker for secreted proteins mediating innate antiviral immunity in vaginal fluids and that drug formulation may impact this activity as reflected in the glycome.

Correlation of rupture dynamics to the nonlinear backscatter response from polymer-shelled ultrasound contrast agents.

Polymer-shelled ultrasound contrast agents (UCAs) may expel their encapsulated gas subject to ultrasound-induced shell buckling or rupture. Nonlinear oscillations of this gas bubble can produce a subharmonic component in the ultrasound backscatter. This study investigated the relationship between this gas-release mechanism and shell-thickness-to-radius ratios (STRRs) of polymer-shelled UCAs. Three types of polylactide-shelled UCAs with STRRs of 7.5, 40, and 100 nm/µm were studied. Each UCA population had a nominal mean diameter of 2 µm. UCAs were subjected to increasing static overpressure ranging from 2 to 330 kPa over a duration of 2 h in a custom-designed test chamber while being imaged using a 200× magnification video microscope at a frame rate of 5 frames/s. Digitized video images were binarized and processed to obtain the cross-sectional area of individual UCAs. Integration of the normalized cross-sectional area over normalized time, defined as buckling factor (Bf), provided a dimensionless parameter for quantifying and comparing the degree of pre-rupture buckling exhibited by the UCAs of different STRRs in response to overpressure. The UCAs with an STRR of 7.5 nm/µm exhibited a distinct shell-buckling phase before shell rupture (Bf < 1), whereas the UCAs with higher STRRs (40 and 100 nm/µm) did not undergo significant prerupture buckling (Bf ≈ 1). The difference in the overpressure response was correlated with the subharmonic response produced by these UCAs. When excited using 20-MHz ultrasound, individual UCAs (N = 3000) in populations that did not exhibit a buckling phase produced a subharmonic response that was an order of magnitude greater than the UCA population with a prominent pre-rupture buckling phase. These results indicate the mechanism of gas expulsion from these UCAs might be a relevant factor in determining the level of subharmonic response in response to high-frequency ultrasound.

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity.

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

Influence of shell properties on high-frequency ultrasound imaging and drug delivery using polymer-shelled microbubbles.

This two-part study investigated shell rupture of ultrasound contrast agents (UCAs) under static overpressure conditions and the subharmonic component from UCAs subjected to 20-MHz tonebursts. Five different polylactide-shelled UCAs with shell-thickness-to-radius ratios (STRRs) of 7.5, 30, 40, 65, and 100 nm/¿m were subjected to static overpressure in a glycerol-filled test chamber. A video microscope imaged the UCAs as pressure varied from 2 to 330 kPa over 90 min. Images were postprocessed to obtain the pressure threshold for rupture and the diameter of individual microbubbles. Backscatter from individual UCAs was investigated by flowing a dilute UCA solution through a wall-less flow phantom placed at the geometric focus of a 20-MHz transducer. UCAs were subjected to 10- and 20-cycle tonebursts of acoustic pressures ranging from 0.3 to 2.3 MPa. A method based on singular-value decomposition (SVD) was employed to obtain a cumulative subharmonic score (SHS). Different UCA types exhibited distinctly different rupture thresholds that were linearly related to their STRR, but uncorrelated with UCA size. The rupture threshold for the UCAs with an STRR = 100 nm/µm was more than 4 times greater than the UCAs with an STRR = 7.5 nm/µm. The polymer-shelled UCAs produced substantial subharmonic response but the subharmonic response to 20- MHz excitation did not correlate with STRRs or UCA-rupture pressures. The 20-cycle excitation resulted in an SHS that was 2 to 3 times that of UCAs excited with 10-cycle tonebursts.