Molecular recognition and proteoglycan mimic arrangement: modulating cisplatin toxicity.

We have demonstrated that cisplatin (CP), an anticancer drug, showed a preference for binding the sulfated-L-iduronic acid (S-L-IdoA) unit over the sulfated-D-glucuronic acid unit of heparan sulfate. The multivalency of S-L-IdoA, such as in the proteoglycan mimic, resulted in distinct modes of cell-surface engineering in normal and cancer cells, with these disparities having a significant impact on CP-mediated toxicity.

High-level production of a novel alginate lyase (FsAly7) from Flammeovirga sp. for efficient production of low viscosity soluble dietary fiber from sodium alginate.

Sodium alginate is one of the most abundant sustainable gum source for dietary fiber production. However, the preparation efficiencies of low viscosity soluble dietary fiber from sodium alginate remain low. Here, a novel alginate lyase gene (FsAly7) from Flammeovirga sp. was identified and high-level expressed in Pichia pastoris for low viscosity soluble dietary fiber production. The highest enzyme production of 3050 U mL-1 was achieved, which is by far the highest yield ever reported. FsAly7 was used for low viscosity soluble dietary fiber production from sodium alginate, and the highest degradation rate of 85.5 % was achieved under a high substrate content of 20 % (w/v). The molecular weight of obtained soluble dietary fiber converged to 10.75 kDa. FsAly7 catalyzed the cleavage of glycosidic bonds in alginate chains with formation of unsaturated non-reducing ends simultaneously in the degradation process, thus altered the chemical structures of hydrolysates. The soluble dietary fiber exhibited excellent properties, including low viscosity, high oil adsorption capacity activity (2.20 ± 0.03 g g-1) and high emulsifying activity (60.05 ± 2.96 mL/100 mL). This investigation may provide a novel alginate lyase catalyst as well as a solution for the efficient production of low viscosity soluble dietary fiber from sodium alginate.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans.

In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.

Wheat derived glucuronokinase as a potential target for regulating ascorbic acid and phytic acid content with increased root length under drought and ABA stresses in Arabidopsis thaliana.

Glucuronokinase (GlcAK) converts glucuronic acid into glucuronic acid-1-phosphate, which is then converted into UDP-glucuronic acid (UDP-GlcA) via myo-inositol oxygenase (MIOX) pathway. UDP-GlcA acts as a precursor in the synthesis of nucleotide-sugar moieties forming cell wall biomass. GlcAK being present at the bifurcation point between UDP-GlcA and ascorbic acid (AsA) biosyntheses, makes it necessary to study its role in plants. In this study, the three homoeologs of GlcAK gene from hexaploid wheat were overexpressed in Arabidopsis thaliana. The GlcAK overexpressing transgenic lines showed decreased contents of AsA and phytic acid (PA) as compared to control plants. Root length and seed germination analyses under abiotic stress (drought and abscisic acid) conditions revealed enhanced root length in transgenic lines as compared to control plants. These results indicate that the MIOX pathway might be contributing towards AsA biosynthesis as evident by the decreased AsA content in the GlcAK overexpressing transgenic Arabidopsis thaliana plants. Findings of the present study will enhance the understanding of the involvement of GlcAK gene in MIOX pathway and subsequent physiological effects in plants.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.

Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

Synthesis of a Systematic 64-Membered Heparan Sulfate Tetrasaccharide Library.

Heparan sulfate (HS) has multifaceted biological activities. To date, no libraries of HS oligosaccharides bearing systematically varied sulfation structures are available owing to the challenges in synthesizing a large number of HS oligosaccharides. To overcome the obstacles and expedite the synthesis, a divergent approach was designed, where 64 HS tetrasaccharides covering all possible structures of 2-O-, 6-O- and N-sulfation with the glucosamine-glucuronic acid-glucosamine-iduronic acid backbone were successfully produced from a single strategically protected tetrasaccharide intermediate. This extensive library helped identify the structural requirements for HS sequences to have strong fibroblast growth factor-2 binding but a weak affinity for platelet factor-4. Such a strategy to separate out these two interactions could lead to new HS-based potential therapeutics without the dangerous adverse effect of heparin-induced thrombocytopenia.

Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa.

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.

Identification of xylan arabinosyl 2-O-xylosyltransferases catalyzing the addition of 2-O-xylosyl residue onto arabinosyl side chains of xylan in grass species.

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

UDP-glucose 6-dehydrogenase lessens sorafenib sensitivity via modulating unfolded protein response.

As the first-generation targeted therapy, sorafenib remains an effective single-drug treatment for advanced hepatocellular carcinoma (HCC). Unfortunately, the existence of resistance restricts the long-term benefit of patients. UDP-glucose 6-dehydrogenase (UGDH) is the key enzyme of glucuronic acid metabolism which was largely reported in mediating drug systemic elimination. In this study, we explore its critical role in regulating sorafenib sensitivity. Here we find sorafenib exposure could activate glucuronic acid metabolism, accompanied with the elevated expression of UGDH. Interference with the route by silencing UGDH could boost HCC cells sensitivity to sorafenib. Meanwhile, the analysis of HCC patients with sorafenib treatment displayed that low UGDH expression predicted superior prognosis. Further screening assay suggested that unfolded protein response (UPR) involves in UGDH silencing-mediated apoptosis. Xenograft model confirmed that combined UGDH intervention could significantly improve sorafenib efficacy. Our results reveal the impact of sorafenib exposure on glucuronic acid metabolism reprogramming and provide UGDH as a promising target to improve sorafenib efficacy.

FRAGILE CULM 18 encodes a UDP-glucuronic acid decarboxylase required for xylan biosynthesis and plant growth in rice.

Although UDP-glucuronic acid decarboxylases (UXSs) have been well studied with regard to catalysing the conversion of UDP-glucuronic acid into UDP-xylose, their biological roles in grasses remain largely unknown. The rice (Oryza sativa) genome contains six UXSs, but none of them has been genetically characterized. Here, we reported on the characterization of a novel rice fragile culm mutant, fc18, which exhibited brittleness with altered cell wall and pleiotropic defects in growth. Map-based cloning and transgenic analyses revealed that the FC18 gene encodes a cytosol-localized OsUXS3 and is widely expressed with higher expression in xylan-rich tissues. Monosaccharide analysis showed that the xylose level was decreased in fc18, and cell wall fraction determinations confirmed that the xylan content in fc18 was lower, suggesting that UDP-xylose from FC18 participates in xylan biosynthesis. Moreover, the fc18 mutant displayed defective cellulose properties, which led to an enhancement in biomass saccharification. Furthermore, expression of genes involved in sugar metabolism and phytohormone signal transduction was largely altered in fc18. Consistent with this, the fc18 mutant exhibited significantly reduced free auxin (indole-3-acetic acid) content and lower expression levels of PIN family genes compared with wild type. Our work reveals the physiological roles of FC18/UXS3 in xylan biosynthesis, cellulose deposition, and plant growth in rice.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Lassa Fever Virus Binds Matriglycan-A Polymer of Alternating Xylose and Glucuronate-On α-Dystroglycan.

Lassa fever virus (LASV) can cause life-threatening hemorrhagic fevers for which there are currently no vaccines or targeted treatments. The late Prof. Stefan Kunz, along with others, showed that the high-affinity host receptor for LASV, and other Old World and clade-C New World mammarenaviruses, is matriglycan-a linear repeating disaccharide of alternating xylose and glucuronic acid that is polymerized uniquely on α-dystroglycan by like-acetylglucosaminyltransferase-1 (LARGE1). Although α-dystroglycan is ubiquitously expressed, LASV preferentially infects vascular endothelia and professional phagocytic cells, which suggests that viral entry requires additional cell-specific factors. In this review, we highlight the work of Stefan Kunz detailing the molecular mechanism of LASV binding and discuss the requirements of receptors, such as tyrosine kinases, for internalization through apoptotic mimicry.

Unique features of the bifunctional GH30 from Thermothelomyces thermophila revealed by structural and mutational studies.

Fungal xylanases belonging to family GH30_7, initially categorized as endo-glucuronoxylanases, are now known to differ both in terms of substrate specificity, as well as mode of action. Recently, TtXyn30A, a GH30_7 xylanase from Thermothelomyces thermophila, was shown to possess dual activity, acting on the xylan backbone in both an endo- and an exo- manner. Here, in an effort to identify the structural characteristics that append these functional properties to the enzyme, we present the biochemical characterization of various TtXyn30A mutants as well as its crystal structure, alone, and in complex with the reaction product. An auxiliary catalytic amino acid has been identified, while it is also shown that glucuronic acid recognition is not mediated by a conserved arginine residue, as shown by previously determined GH30 structures.

Biochemical Characterization of a Novel Exo-Type PL7 Alginate Lyase VsAly7D from Marine Vibrio sp. QY108.

Brown algae is a kind of renewable resource for biofuels production. As the major component of carbohydrate in the cell walls of brown algae, alginate can be degraded into unsaturated monosaccharide by exo-type alginate lyases, then converted into 4-deoxy-L-erythro-5-hexoseulose uronate (DEH) by a non-enzyme reaction, which is an important raw material for the preparation of bioethanol. In our research, a novel exo-type alginate lyase, VsAly7D, belonging to the PL7 family was isolated from marine bacterium Vibrio sp. QY108 and recombinantly expressed in Escherichia coli. The purified VsAly7D demonstrated the highest activity at 35 °C, whereas it still maintained 46.5% and 83.1% of its initial activity at 20 °C and 30 °C, respectively. In addition, VsAly7D exhibited the maximum activity under alkaline conditions (pH 8.0), with the simultaneously remaining stability between pH 8.0 and 10.0. Compared with other reported exo-type enzymes, VsAly7D could efficiently degrade alginate, poly-ß-D-mannuronate (polyM) and poly-α-L-guluronate (polyG) with highest specific activities (663.0 U/mg, 913.6 U/mg and 894.4 U/mg, respectively). These results showed that recombinant VsAly7D is a suitable tool enzyme for unsaturated alginate monosaccharide preparation and holds great promise for producing bioethanol from brown algae.

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family.

Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the ß-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1→4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1→4)-d-glucuronic acid -bound FoRham1 were determined, and the active site was identified on the anterior side of the ß-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.

Carbon dots-embedded N-acetylneuraminic acid and glucuronic acid-imprinted polymers for targeting and imaging of cancer cells.

Diagnosis, treatment, and prediction of cancer progression require new targeting agents to specifically target cell surface receptors. Herein, we demonstrated fluorescent carbon quantum dots-molecularly imprinted polymer (CQD-MIP) for selective targeting and imaging of cancer cells. Carbon quantum dots (CQDs) were synthesized and characterized. The synthesized CQDs had average size of 1.5 nm and show intense fluorescence emission at wavelength of 450 nm with excitation at 370 nm. CQD-MIP nanoparticles imprinted with N-acetylneuraminic acid and glucuronic acid were prepared and characterized. CQD-MIPs were successfully applied for selective targeting and imaging of MCF-7, HepG-2, and NIH-3T3 cell lines. Non-imprinted polymer (NIP) showed no binding properties toward a target molecule. Non-imprinted polymer (NIP) and non-cancerous human cell lines were used for controlling the imprinting and targeting effects, respectively. Acceptable results were obtained with imprinted polymers on cancer cells.

Analysis of the biosynthetic flux in bovine milk oligosaccharides reveals competition between sulfated and sialylated species and the existence of glucuronic acid-containing analogues.

We previously observed that sialylated bovine milk oligosaccharides (BMOs) decline in both absolute and relative abundances over the initial stages of bovine lactation, with initial evidence suggesting that this decline occurred due to increased concentrations of unique sulfated BMOs. Since both sulfated and sialylated BMOs have distinct bioactivites, a follow up study was launched in order to more clearly define relative changes in these classes of BMOs over the first week of lactation in dairy cattle. Capillary electrophoresis (CE) and several liquid chromatography mass spectrometry (LC-MS) methods, including a novel multiplexed tandem MS method, were used to profile the BMOs extracted from milk collected from the same 20 Holstein cows at milkings 1, 2, 3, 4, 8, and 14 post-partum. In addition to clearly validating that sulfated and sialylated BMOs exist in direct biosynthetic completion, our study has identified over 170 unique BMOs including 14 unique glucuronic acid-containing trisaccharides.