Biosynthesis and intracellular pool of aminopeptidase N in rabbit enterocytes

Summary A papain treatment at 15°C and pH 7.3 of a microsomal fraction from rabbit enterocytes quantitatively releases the aminopeptidase N integrated in the plasma membranes without solubilizing the enzyme integrated in the intracellular membranes. Working on A⁺ rabbits, characterized by the presence on the brush-border hydrolases of glycans corresponding to the human blood group A-determinant structure, it was possible to separate the intracellular aminopeptidase into two major molecular forms with or without these determinants. The molecular form devoid of human blood group A antigenicity corresponds to the only stable intermediate of glycosylation, bearing N-linked high mannose oligosaccharides. This endoglycosidase H-sensitive form is fully active and represents in the steady state about 1% of the total cellular aminopeptidase. It contains a cytoplasmic sequence of about 3000 daltons that has not yet been detected in the mature form. The A antigenicity is acquired simultaneously with processing of high mannose glycans to complex glycans. Pulse chase labeling of jejunum loops with [³⁵S]-methionine showed that the complete processing of the transient form synthesized during 10 min takes 1 hr. During the last 30 min of processing, all the newly transformed molecules are transported to the plasma membrane.     

Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV.

The temporal association between O-glycosylation and processing of N-linked glycans in the Golgi apparatus as well as the implication of these events in the polarized sorting of three brush border proteins has been the subject of the current investigation. O-Glycosylation of pro-sucrase-isomaltase (pro-SI), aminopeptidase N (ApN), and dipeptidyl peptidase IV (DPPIV) is drastically reduced when processing of the mannose-rich N-linked glycans is blocked by deoxymannojirimycin, an inhibitor of the Golgi-located mannosidase I. By contrast, O-glycosylation is not affected in the presence of swainsonine, an inhibitor of Golgi mannosidase II. The results indicate that removal of the outermost mannose residues by mannosidase I from the mannose-rich N-linked glycans is required before O-glycosylation can ensue. On the other hand, subsequent mannose residues in the core chain impose no sterical constraints on the progression of O-glycosylation. Reduction or modification of N- and O-glycosylation do not affect the transport of pro-SI, ApN, or DPPIV to the cell surface per se. However, the polarized sorting of two of these proteins, pro-SI and DPPIV, to the apical membrane is substantially altered when O-glycans are not completely processed, while the sorting of ApN is not affected. The processing of N-linked glycans, on the other hand, has no influence on sorting of all three proteins. The results indicate that O-linked carbohydrates are at least a part of the sorting mechanism of pro-SI and DPPIV. The sorting of ApN implicates neither O-linked nor N-linked glycans and is driven most likely by carbohydrate-independent mechanisms.     

Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N.

The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo-beta-N-acetylglucosaminidase H. Swainsonine caused only a moderate inhibition of transport of the enzyme through the Golgi complex and the subsequent expression in the microvillar membrane. This may imply that the trimming of the high-mannose core and complex glycosylation of N-linked oligosaccharides is not essential for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment of the Mr-160000 polypeptide with endo-beta-N-acetylglucosaminidase H only decreased its apparent Mr by 15000. The susceptibility of the mature Mr-166000 polypeptide, but not the Mr-140000 polypeptide, to mild alkaline hydrolysis suggests that aminopeptidase N becomes glycosylated with O-linked oligosaccharides during its passage through the Golgi complex. Aminopeptidase N was not labelled by [3H]palmitic acid, indicating that the processing of the enzyme does not include acylation.     

Selective organic precipitation/extraction of released N-glycans following large-scale enzymatic deglycosylation of glycoproteins

A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N(4)-(N-acetyl-beta-glucosaminyl) Asn amidase (EC 3.5.1. 52)-released complex, and beta-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at -20 degrees C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.     

Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells

The carboxyl-terminal fibrin-binding domain, Fib2, of human fibronectin was expressed in mouse L cells as a fusion protein with the signal sequence of human protein C inhibitor. The recombinant Fib2 (rFib2) protein synthesized by transfected cells retained the ability to form dimers with each other or with mouse fibronectin subunits and was secreted to the medium after extensive glycosylation. Only a small fraction of the secreted protein was incorporated into the pericellular matrix. Interestingly, the secreted rFib2 protein displayed a remarkable heterogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, giving rise to a broad band corresponding to Mr of 60,000-90,000. The heterogeneity was eliminated mostly by treatment with neuraminidase and further by treatment with endo-alpha-N-acetylgalactosaminidase. Treatment with peptide:N-glycosidase F did not alter the heterogeneity of the protein, indicating that differential sialylation of O-linked, but not N-linked, glycans is largely responsible for the apparent heterogeneity. The presence of O-linked but absence of N-linked glycans was further supported by the observations that peanut agglutinin specifically bound to the desialylated rFib2 protein, whereas neither concanavalin A nor lentil lectin bound to the protein irrespective of prior neuraminidase treatment. Since the apparent heterogeneity of the rFib2 protein was only observable with the secreted, but not the cytoplasmic form, sialylation of O-linked glycans may be essential for, or regulate as a rate-limiting step, the transit of the recombinant protein to the extracellular space.     

Analysis of protein glycosylation by mass spectrometry

We present a detailed protocol for the structural analysis of protein-linked glycans. In this approach, appropriate for glycomics studies, N-linked glycans are released using peptide N-glycosidase F and O-linked glycans are released by reductive alkaline beta-elimination. Using strategies based on mass spectrometry (matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS-MS)), chemical derivatization, sequential exoglycosidase digestions and linkage analysis, the structures of the N- and/or O-glycans are defined. This approach can be used to study the glycosylation of isolated complex glycoproteins or of numerous glycoproteins encountered in a complex biological medium (cells, tissues and physiological fluids).     

Structure determination of the major N- and O-linked carbohydrate chains of the beta subunit from equine chorionic gonadotropin

The carbohydrate moieties of equine chorionic gonadotropin alpha and beta subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the beta subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly alpha 1-6 fucosylated at the Asn-bound GlcNAc residue and having alpha 2-6 and alpha 2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the alpha subunit. The major part of the O-linked carbohydrate chains, occurring solely in the beta subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the beta subunit.     

Structure, biosynthesis, and glycosylation of human small intestinal maltase-glucoamylase

Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential glycosylation produces heterogeneity in elevated esterases associated with insecticide resistance in the brown planthopper Nilaparvata lugens Stål

The major insecticide resistance mechanism in the brown planthopper Nilaparvata lugens involves overproduction of esterases. Esterases purified from a resistant strain appeared as a ladder of bands on isoelectric focussing (IEF) gels from pI 4.7 to 5.0. Two-dimensional electrophoresis showed that isozymes ranged in size from 66 to 68 kDa with those of lower pI being apparently smaller. All isozymes detected by two-dimensional electrophoresis were glycosylated. N-glycosidase A reduced the number of isozymes on IEF to two, with increased pI and an increased molecular weight of 69 kDa. No O-linked glycans were detected. Deglycosylation had no effect on esterase activity, hence glycosylation is not involved in active site conformation. As N-glycosidase F completely deglycosylated the esterases, none of the glycans has an alpha1,3-bound core fucose. Reactivity with the lectins GNA, MAA and DSA, combined with differential cleavage of N-linked glycans with endoglycosidases F1 and F2, indicated that terminally linked mannose is present in high mannose and/or hybrid type glycans and that terminally linked sialic acid and galactose-beta(1-4)-N-acetylglucosamine are present in biantennary complexes. Neuraminidase treatment had the same effect on pI of isozymes as complete deglycosylation. Therefore, the majority of the heterogeneity of elevated esterases on IEF is due to differential attachment of sialic acid to glycans of the two proteins.     

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ～30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.     

Facile removal of high mannose structures prior to extracting complex type N‐glycans from de‐N‐glycosylated peptides retained by C18 solid phase to allow more efficient glycomic mapping

The relative amount of high mannose structures within an N‐glycomic pool differs from one source to another, but quite often it predominates over the larger size complex type structures carrying biologically important glyco‐epitopes. An efficient method to separate these two classes of N‐glycans would significantly aid in detecting the lower abundant components by MS. Capitalizing on an initial observation that only high mannose type structures were recovered in the flow‐through fraction when peptide‐N‐glycosidase F digested peptides were passed through a C18 cartridge in 0.1% formic acid, we demonstrated here that native complex type N‐glycans can be retained by C18 cartridge and to be efficiently separated from both the smaller high mannose type structures, as well as de‐N‐glycosylated peptides by stepwise elution with increasing ACN concentration. The weak retention of the largely hydrophilic N‐glycans on C18 resin is dependent not only on size but also increased by the presence of α6‐fucosylation. This was shown by comparing the resulting N‐glycomic profiles of the washed and low‐ACN eluted fractions derived from both a human cancer cell line and an insect cell line.     

The carbohydrate moiety of α-galactosidase from Trichoderma reesei

a-Galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by b-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: a1-6 D-mannopyranosyl- a-D-glycopyranoside and a1-6 D-glucopyranosyl- a-D-galactopyranoside and one trimer: a-D-glucopyranosyl- a1-2 D-mannopyranosyl- a1-6 D-galac-topyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo- b-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo- b- N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of a-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of a-glactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assembly and processing of the disulfide-linked varicella-zoster virus glycoprotein gpII(140).

Varicella-zoster virus (VZV) specifies the synthesis of at least four families of glycoproteins, which have been designated gpI, gpII, gpIII, and gpIV. In this report we describe the assembly and processing of VZV gpII, a structural protein of an apparent Mr of 140,000, which is the homolog of gB of herpes simplex virus. For these studies, we used two anti-gpII monoclonal antibodies which exhibited both complement-independent neutralization activity and inhibition of virus-induced cell-to-cell fusion. Pulse-chase labeling experiments identified a 124,000-Mr intermediate which was chased to the mature 140,000-Mr product when analyzed in nonreducing gels; in the presence of a reducing agent, the native gp140 was cleaved into two closely migrating species (gp66 and gp68). The biosynthesis of VZV gpII was further analyzed in the presence of the following inhibitors of glycoprotein processing: tunicamycin, monensin, castanospermine, swainsonine, and deoxymannojirimycin. All intermediate and mature forms were digested with endoglycosidases H and F, neuraminidase, and O-glycanase to further define high-mannose, complex, and O-linked glycans. Finally, the addition of sulfate residues was investigated. This characterization of VZV gpII revealed the following results. (i) gp128 and gp124 were early high-mannose forms, (ii) gp126 was an intermediate form with complex N-linked oligosaccharides, (iii) gp130 was a later intermediate with both N-linked and O-linked glycans, and (iv) the mature product gp140 contained a mixture of N-linked and O-linked glycans which were both sialated and sulfated. Further investigations indicated that gpII sulfation was inhibited by tunicamycin and castanospermine but not by deoxymannojirimycin or swainsonine. We also concluded that VZV gpII displayed many biological and biochemical properties similar to those of its herpes simplex virus homolog gB.     

Glycosylation of proteins in plants and invertebrates

N-glycosylation is the most conserved form of protein glycosylation in eukaryotes, but the modifications of N-linked oligosaccharides in plants and invertebrates often differ greatly from those in vertebrates and sometimes result in immunogenic structures. By contrast, O-linked glycans tend to be a wide and disparate group of modifications. Whereas the forms of O-linked glycans in plants are unlike those in animals, studies on invertebrate O-glycosylation often yield information relevant to mammalian systems.     

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

Glycan structures of ocular surface mucins in man,rabbit and dog display species differences

The composition of the mucus gel of the tear film reflects the competing needs for transparency, stability, hydration, and protection of the ocular surface. Mucins form the macromolecular scaffolding of this hydrated gel, and glycans decorating these glycoproteins represent a rich source of binding ligands that may both modulate microbial binding and regulate the physicochemical characteristics of the gel. This study compares the structure of O-linked glycans derived from the ocular mucins of three species, to determine whether the ocular surface microenvironment dictates the need for a common pattern of O-linked carbohydrate structures. Ocular mucus aspirates were collected from healthy humans, rabbits and dogs. Mucins were purified using standard protocols. O-glycans were released by hydrazinoloysis and subsequently analysed by a combination of HPLC, exoglycosidase digestions and LC–MS/MS. A total of 12 different O-glycans were identified. In human ocular mucin, the majority were negatively charged and terminated in sialic acid, whilst those from rabbit or dog were mainly neutral and terminated in α 1-2 fucose and/or α 1-3 N-acetylgalactosamine. The glycans were short: the most common structures being tetra-, tri- or disaccharides. Less elaborate glycan structures are encountered at the ocular surface than at many other mucosal surfaces. Species-specific glycan expression is a feature of ocular surface mucins, and has implications for their defensive properties where different microbial and environmental challenges are encountered. Louise Royle and Elizabeth Matthews contributed equally to this work.     

Purification and characterization of a lectin from endophytic fungus Fusarium solani having complex sugar specificity

A lectin from the mycelial extract of an endophytic strain of Fusarium solani was purified. Its hemagglutinating activity was inhibited by glycoproteins possessing N-linked as well as O-linked glycans. The thermodynamics and kinetics of binding of glycans and glycoproteins to F. solani lectin was studied using surface plasmon resonance. The lectin showed high affinity for asialofetuin, asialomucin, asialofibrinogen, and thyroglobulin; and comparatively low affinity for mucin, fetuin, fibrinogen, and holotransferrin. Glycoproteins showed several fold higher affinity than their corresponding glycans with significant contribution from enthalpy and positive entropy, suggesting the involvement of non-polar protein-protein interaction. Moreover, the higher affinity of the glycoproteins was due to their faster association rates and low activation energy.     

基于FFPE组织切片的膀胱癌N-连接糖链原位酶解及分析

目的 研究膀胱癌FFPE组织切片的N-连接糖链,发现膀胱癌FFPE肿瘤组织的异常N-连接糖链修饰情况。方法 发展基于FFPE组织切片原位提取N-连接糖链的实验流程。通过PNGase F酶切FFPE组织解释放N-连接糖链。对N-连接糖链自由端进行全甲基化修饰。通过MALDI-TOF/TOF-MS检测N-连接糖链的相对含量。进行数据库匹配,确定N-连接糖链的可能糖型。ROC分析用于预测显著差异N-连接糖链作为预测膀胱癌生物标志物的准确度。结果 MALDI-TOF/TOF-MS检测泛甲基化修饰N-连接糖链的数据显示,在16例膀胱癌患者的肿瘤和癌旁组织的3次重复实验中,肿瘤组织中蛋白质高甘露糖型N2H6、N2H7、N2H8、N2H9和复杂型N5H6F1糖链修饰水平显著上升,同时高甘露糖型N2H5、杂合型N3H5以及复杂型N3H4、N4H4、N5H6F1S2糖链修饰水平显著下降。ROC分析显示,双天线型N-连接糖链N3H4（AUC=0.90）和N4H4（AUC=0.91）在单独或者共同区分膀胱癌患者肿瘤组织和癌旁组织中都具有很好的可靠性,可能成为膀胱癌的潜在生物标志物。结论 膀胱癌FFPE肿瘤组织中存在蛋白质异常N-糖基化修饰,N-连接糖链N3H4和N4H4或可成为膀胱癌的潜在生物标志物。     

Abnormal Galactosylated–Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.