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Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Highlights • FACE is a simple, qualitative and quantitative method. • The standard curve warrants quantification of chitooligosaccharides of up to 10 nmol regardless on used buffer system. • Our improved FACE method enable us to quantify chitooligosaccharides produced by chitinases at pH 2.0–8.0. • Determination of the combinatory effects of the Chit1 and AMCase using the FACE method., Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of degradation products from artificial and natural chitin substrates such as 4-NP-(GlcNAc)2, (GlcNAc)4 and colloidal chitin. The FACE method has been improved by our group for analysis of chitooligosaccharides in the presence of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities of enzymes at pH 2.0–8.0. FACE is a very sensitive technique detecting picomolar amounts of molecules. We optimized the detection conditions as follows: exposure type, precision; sensitivity, high resolution; exposure time, 5 s. We evaluated the (GlcNAc)2 levels using a standard curve that allows chitooligosaccharides quantification at up to 10 nmol amounts. Using the method presented here, the chitinolytic properties of different chitinases can be compared directly. Serratia chitinase A (ChiA) and chitinase B (ChiB), two well-studied bacterial chitinases, have been shown by HPLC to have a synergistic effect on the chitin degradation rate. Using the FACE method, we determined the combinatory effects of mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) in natural chitin substrates processing.•FACE is a simple and quantitative method.•Our improved procedure enables the quantification of chitooligosaccharides produced by chitinases at pH 2.0–8.0.•FACE is able to quantify chitooligosaccharides at up to 10 nmol amounts., Graphical abstract Image, graphical abstract

DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis allows for accurate profiling of the asparagine-linked (N-) glycosylation patterns, a posttranslational modification present on many soluble and membrane proteins. This technique has been extensively tested to identify N-glycosylation patterns associated with serum proteins. Here we describe the use of DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis to identify the N-glycosylation patterns of prostate proteins in urine.

Graphical abstract, The building blocks of simple and complex oligosaccharides, termed sugar nucleotides, are often overlooked for their role in metabolic diseases and may hold the key to the underlying disease pathogenesis. Multiple reasons may account for the lack of analysis and quantitation of these sugar nucleotides, including the difficulty in isolation and purification as well as the required expensive instrumentation such as a high performance liquid chromatography (HPLC), mass spectrometer, or capillary electrophoresis. We have established a simple yet effective way to purify and quantitate sugar nucleotides using solid phase extraction (SPE) chromatography combined with fluorophore assisted carbohydrate electrophoresis (FACE). The simplicity of use, combined with the ability to run multiple samples at one time, give this technique a distinct advantage over the established methods for isolation and analysis of sugar nucleotides from cell culture models. • Sugar nucleotides can be easily purified with solid phase extraction chromatography. • FACE can be used to analyze multiple nucleotide sugar extracts with a single run. • The proposed method is simple, affordable, and uses common everyday research labware.

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a method in which a fluorophore is covalently attached to the reducing end of carbohydrates, thereby allowing visualization following high-resolution separation by electrophoresis. This method can be used for carbohydrate profiling and sequencing, as well as for the determination of the specificity of carbohydrate-active enzymes. Here, we describe and demonstrate the use of FACE to separate and visualize the glycans released following digestion of oligosaccharides by glycoside hydrolases (GHs) using two examples: (1) the digestion of chitobiose by the streptococcal β-hexosaminidase GH20C, and (2) the digestion of glycogen by the GH13 member SpuA.

Fluorophore-assisted carbohydrate electrophoresis (FACE) was applied to determine the molecular mass (M) values of various chondroitin sulfate (CS) samples. After labeling with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), FACE was able to resolve each CS sample as a discrete band depending on the M value. After densitometric acquisition, the migration distance of each CS standard was acquired and the third grade polynomial calibration standard curve was determined by plotting the logarithms of the M values as a function of migration ratio. Purified CS samples of different origin and the European Pharmacopeia CS standard were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (Mp), the number-average Mn, weight-average Mw, and polydispersity (Mw/Mn) were examined by both techniques and found to be quite similar. This study demonstrates that FACE analysis is a suitable, sensitive and simple method for the determination of the M values of CS macromolecules with possible utilization in virtually any kind of research and development such as quality control laboratories.

The injectable and hydrophilic nature of hydrogels makes them suitable candidates for cartilage tissue engineering. To date, a wide range of hydrogels have been proposed for articular cartilage regeneration but few studies have quantitatively compared chondrocyte behaviour and extracellular matrix (ECM) synthesis within the hydrogels. Herein we have examined the nature of ECM synthesis by chondrocytes seeded into four hydrogels formed by either temperature change, self-assembly or chemical cross-linking. Bovine articular cartilage chondrocytes were cultured for 14 days in Extracel, Pluronic F127 blended with Type II collagen, Puramatrix and Matrixhyal. The discriminatory and sensitive technique of fluorophore-assisted carbohydrate electrophoresis (FACE) was used to determine the fine detail of the glycosaminoglycans (GAG); hyaluronan and chondroitin sulphate. FACE analysis for chondroitin sulphate and hyaluronan profiles in Puramatrix closely matched that of native cartilage. For each hydrogel, DNA content, viability and morphology were assessed. Total collagen and total sulphated GAG production were measured and normalised to DNA content. Significant differences were found in total collagen synthesis. By day 14, Extracel and Puramatrix had significantly more total collagen than Matrixhyal (1.77+/-0.26 microg and 1.97+/-0.26 microg vs. 0.60+/-0.26 microg; p

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straight-forward, sensitive method for determining the presence and relative abundance of individual (oligo) saccharide in a(n) (oligo) saccharide mixture. The single terminal aldehydes of (oligo) saccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling is not biased by (oligo) saccharide length. Therefore, band fluorescence intensity is directly related to the relative abundance of individual (oligo) saccharide moieties in heterogeneous sample. In the same time, it also indicates that FACE can be used to investigate the interactions of carbohydrates and proteins.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual (oligo)saccharide in a(n) (oligo)saccharide mixture. The single terminal aldehydes of oligoglucoside residues released by acid hydrolysis of β-1, 3-d-glucan from yeast were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligoglucoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligoglucoside moieties in heterogeneous sample. Therefore, FACE represents an accessible, sensitive, and quantitative analytical tool enabling the analysis of (oligo)saccharides derived from different sources.

A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.

An inexpensive, fast, simple, sensitive and reproducible fluorophore-assisted carbohydrate electrophoresis (FACE) method is described for the quantitative analysis of microgram amounts of the Escherichia coli K4 bacterium capsule polysaccharide and its defructosylated polymer. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, ΔHexAFru-GalNAc for K4 and ΔHexA-GalNAc for defructosylated K4, are fluorotagged by 2-aminoacridone (AMAC) and the products separated on a polyacrylamide gel electrophoresis. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from 0.5 to 5 μg for the Δ-disaccharide of K4 and from 0.2 to 5 μg for the Δ-disaccharide of K4d. The detection limit was found to be approx. 0.5–1 μg for K4 and 0.1–0.2 μg for K4d. The FACE procedure described is especially useful when many samples need to be analyzed.

Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [(3)H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [(3)H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [(3)H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc(3)Man(9)GlcNAc(2)-P-P-dolichol, without hypoglycosylation, CDG-Ia fibroblasts grown with physiological glucose. This suggested a "missing link" to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This led to futile cycling the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.

Lipid-linked oligosaccharides (LLOs) such as Glc3Man9GlcNAc2-P-P-dolichol are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I Congenital Disorders of Glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [3H]mannose or [14C]glucosamine. In this article, a procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO analysis based on fluorophore-assisted carbohydrate electrophoresis (FACE). First, LLOs are extracted and partially purified. Next, oligosaccharides released from LLOs are labeled with negatively charged fluorophores: 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) or 7-amino-1,3-naphthalenedisulfonic acid (ANDS). A specialized form of polyacrylamide gel electrophoresis is then used to resolve and measure ANTS or ANDS labeled oligosaccharides. Finally, the resolved oligosaccharides are detected and quantified by fluorescence imagers using CCD cameras.

Fluorophore‐assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual (oligo)saccharide in a(n) (oligo)saccharide mixture. The single‐terminal aldehydes of oligoglucoside residues released by acid hydrolysis of β‐1,3 glucan from yeast were tagged with the charged fluorophore 8‐aminonaphthalene‐1,3,6‐trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligoglucoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligoglucoside moieties in heterogeneous samples. FACE analysis revealed that the major oligoglucoside mixture released by acid hydrolysis from β‐1,3 glucan was composed of monosaccharide, disaccharide, trisaccharide, and tetrasaccharide, respectively. And the best hydrolytic condition is at a concentration of 2.0 mol/L TFA, heated a...

In this report, we describe a polyacrylamide gel electrophoresis for the analysis of fluorophore-labelled hyaluronan (HA) and chondroitin sulfate (CS) Δ-disaccharides. The method utilizes derivatization of reducing ends of hyaluronan and the variously sulfated chondroitin sulfate Δ-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis in Tris–HCl buffer (pH 8.8), and polyacrylamide gel (T 25%/C 3.75%). The method was applied to the analysis of GAGs secreted into the culture medium of human aortic smooth muscle cells and the obtained results were compared with those analysed by fluorophore assisted carbohydrate electrophoresis (FACE). The described method is a useful and sensitive tool for the rapid monitoring of GAGs in high number of biologic samples.

The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.

Explanted porcine bioprosthetic valves have a thinned spongiosa, partially because of an overall loss of glycosaminoglycans (GAGs). We measured the concentrations of specific GAG classes in explanted bioprosthetic valves (n = 14, implanted 12.0 ± 4.7 years) compared with glutaraldehyde-fixed porcine controls. After extraction with NaOH, GAGs were analyzed using either a hexuronic acid assay or fluorophore-assisted carbohydrate electrophoresis to quantify the individual GAG classes. The total GAG concentration in explants was 198 ± 95 pmol/mg wet weight—93% less than freshly fixed controls. Explants also contained altered proportions of the different GAG classes relative to controls. The proportions of hyaluronan and chondroitin/dermatan-6-sulfate were reduced from 39 to 7% and 34 to 18% of total GAGs, respectively. The predominant explant GAG class was chondroitin/dermatan-4-sulfate (proportion elevated from 14 to 70%). This GAG is commonly found in the collagen-associated proteoglycan decorin, which is likely well crosslinked by glutaraldehyde. Chondroitin-6-sulfate is commonly found in the water- and hyaluronan-binding proteoglycan versican, which is likely poorly crosslinked. The loss of versican and its associated water-binding capacity is consistent with the thinned spongiosa. The resultant compromise of hydration, compressive resistance, and viscoelasticity may be responsible for the deterioration of the bioprosthesis in vivo. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 251–259, 2003

Saccharides were evaluated as biomarkers for cadmium (Cd) and starvation using fluorophore-assisted carbohydrate-electrophoresis (FACE) in 4th instar Chironomus riparius . FACE allowed different types of saccharides in whole larval homogenate to be analyzed simultaneously and in parallel with other larval samples. Larval homogenates showed seven principle bands labeled A, B, C, D, E, F and G. Previous work found that the migration patterns of bands A, C, D and F matched those of ribose, glucose, galactose and fructose, respectively. Four of the bands, B, C, E and G were generated from glucose-based mono, oligo, and polysaccharides. Band B was primarily derived from glucose and band E from glycogen. Experiments (0–72 h) with starved larvae showed a time dependent reduction in bands B and E that was statistically significant at 72 h. Experiments with Cd (0.01–1000 μM) showed a concentration and time dependent reduction in band E with a LOEL of 1 μM and NOEL of 0.01 μM at 48 h. The LOEL was 0.014% of the 48 h LC50. Significant reduction of band E only occurred in fed larvae indicating that food was an important route of exposure. Reductions in saccharides were independent of larval weight loss at 48 h. This suggested that major changes in saccharides were not due to weight loss but metabolic stress in the presence of Cd.

Hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS) are glycosaminoglycans (GAGs) with a great importance in biological processes as they participate in functional cell properties, such as migration, adhesion, and proliferation. A perturbation of the quantity and/or the sulfation of GAGs is often associated with pathological conditions. In this chapter, we present valuable and validated protocols for the analysis of HA-, CS-, and HS-derived disaccharides after derivatization with 2-aminoacridone and by using the fluorophore-assisted carbohydrate electrophoresis (FACE). FACE is a well-known technique and a reliable tool for a fast screening of GAGs, as it is possible to analyze 16 samples at the same time with one electrophoretic apparatus. The protocols for the gel preparation are based on the variations of the acrylamide/bisacrylamide and buffer concentrations. Different approaches for the extraction and purification of the disaccharides of various biologic samples and pharmaceutical preparations are also stressed.

Glycosaminoglycans (GAGs) due to their hydrophilic character and high anionic charge densities play important roles in various (patho)physiological processes. The identification and quantification of GAGs in biological samples and tissues could be useful prognostic and diagnostic tools in pathological conditions. Despite the noteworthy progress in the development of sensitive and accurate methodologies for the determination of GAGs, there is a significant lack in methodologies regarding sample preparation and reliable fast analysis methods enabling the simultaneous analysis of several biological samples. In this report, developed protocols for the isolation of GAGs in biological samples were applied to analyze various sulfated chondroitin sulfate- and hyaluronan-derived disaccharides using fluorophore-assisted carbohydrate electrophoresis (FACE). Applications to biologic samples of clinical importance include blood serum, lens capsule tissue and urine. The sample preparation protocol followed by FACE analysis allows quantification with an optimal linearity over the concentration range 1.0–220.0 µg/mL, affording a limit of quantitation of 50 ng of disaccharides. Validation of FACE results was performed by capillary electrophoresis and high performance liquid chromatography techniques.

Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.

Lubrication of mammalian joints is mediated by lubricin, a product of megakaryocyte stimulating factor gene (MSF; GenBank accession #U70136) expression. Lubricin (Mr ∼ 240 kDa) is a mucinous glycoprotein which is 50% (w/w) post-translationally modified with β(1-3)Gal-GalNAc incompletely capped with NeuAc, and lubricates apposed cartilaginous surfaces in the boundary mode through an unknown mechanism. Both bovine and human lubricin were purified from synovial fluid and digested with recombinant glycosidases. Released oligosaccharides were identified and quantified by fluorophore assisted carbohydrate electrophoresis (FACE). Corresponding digests of human lubricin were also assayed in a friction apparatus oscillating latex rubber against polished glass at a pressure of 0.35 × 106 N/m2 and the coefficient of friction (μ) was measured. Digestion with α2,3-neuraminidase decreased lubricating ability by 19.3%. Partial removal of β(1-3)Gal-GalNAc moieties by endo-α-N-acetyl-D-galactosaminidase reduced lubricating ability by 77.2%. Human lubricin digested with combined α2,3-neuraminidase and β1-3,6-galactosidase continued to lubricate at 52.2% of its nominal value. Both bovine and human lubricin released 48.6% and 54.4% of total β(1-3)Gal-GalNAc sidechains following digestion with endo-α-N-acetyl-D-galactosaminidase. Biological boundary lubrication by synovial fluid in vitro is provided primarily by extensive O-linked β(1-3)Gal-GalNAc.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual oligomannosyl residues in Candida mannoprotein, the major antigenic determinant located on the outer surface of the yeast cell wall. The single terminal aldehydes of oligomannosyl residues released by hydrolysis were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligomannoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligomannoside moieties in heterogeneous samples. FACE analysis revealed the major oligomannosides released by acid hydrolysis and β-elimination of Fehling-precipitated mannan from Candida albicans , which were the same as those previously reported in studies based on mass and nuclear magnetic spectroscopic analysis. FACE was also amenable to the analysis of samples obtained by direct hydrolysis of whole yeast cells. Whole-cell acid hydrolysis and whole-cell β-elimination of two isolates each of C. albicans , C. glabrata , C. krusei , C. lusitaniae , C. parapsilosis , C. rugosa , C. stellatoidea , and C. tropicalis resulted in oligomannoside gel banding patterns that were species and strain specific for the 16 isolates surveyed. Whereas some bands were specific for an individual isolate or species, other bands were shared by two or three species in various groupings. Differences in the mannoprotein composition of C. albicans A9 and four spontaneous cell surface mutants were also detected. Mannan “fingerprints,” or banding pattern profiles, derived from the electrophoretic mobilities of individual bands relative to the migration of acid-hydrolyzed dextran (relative migration index) yielded profiles characteristic of individual isolates not revealed by standard assimilation and biochemical profiles. FACE represents an accessible, sensitive, and quantitative analytical tool enabling the characterization of yeast mannan complexity.