N-Glycosylation profiling of recombinant mouse extracellular superoxide dismutase produced in Chinese hamster ovary cells

Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans.     

Misfolding of glycoproteins is a prerequisite for peptide: N-glycanase mediated deglycosylation

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the beta-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.     

The structural basis of the difference in sensitivity for PNGase F in the de-N-glycosylation of the native bovine pancreatic ribonucleases B and BS

In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide- N (4)-( N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH-CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21-124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase B (cm)). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase B (cm). NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the alpha-helix of the amino acid residues 1-20 has a stabilizing effect. In RNase BS, the alpha-helix formed by amino acid residues 23-32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS-PNGase F complex is proposed.     

Unique peptide:N-glycanase of Caenorhabditis elegans has activity of protein disulphide reductase as well as of deglycosylation

Peptide:N-glycanase (PNGase) is the enzyme responsible for de-N-glycosylation of misfolded glycoproteins in the cytosol. Here, we report the molecular identification and characterization of PNGase (png-1, F56G4.5) from Caenorhabditis elegans. This enzyme released both high mannose- and complex-type N-glycans from glycopeptides and denatured glycoproteins. Deglycosylation activity was inhibited by Zn(2+) and z-VAD-fmk, but not by EDTA. PNG-1 has a thioredoxin-like domain in addition to a transglutaminase domain, the core domain of PNGases, and exhibited protein disulphide reductase activity in vitro. Our biochemical studies revealed that PNG-1 is a unique bifunctional protein possessing two enzyme activities.     

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.     

Direct on-membrane glycoproteomic approach using MALDI-TOF mass spectrometry coupled with microdispensing of multiple enzymes

We report a novel approach for direct on-membrane glycoproteomics by digestion of membrane-blotted glycoproteins with multiple enzymes using piezoelectric chemical inkjet printing technology and on-membrane direct MALDI-TOF mass spectrometry. With this approach, both N-linked glycan analyses and peptide mass fingerprinting of several standard glycoproteins were successfully performed using PNGase F and trypsin microscale digestions of the blotted spots on membrane from an SDS-PAGE gel. In addition, we performed a similar analysis for 2-DE separated serum glycoproteins as a demonstration of how the system could be used in human plasma glycoproteomics.     

Comparative proteomic analysis of de-N-glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status

Analysis of the polypeptide profile in tissues, cells, and sera by high-resolution two-dimensional (2-D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N-linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de-N-glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de-N-glycosylation by incubation with PNGase F and resolved by high-resolution 2-D gel electrophoresis. De-N-glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de-N-glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de-N-glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.     

利用N糖苷酶F对单克隆抗体N糖酶解条件的优化

为了优化利用N糖苷酶F（PNGase F）酶解单克隆抗体中N糖的方法，应用本公司生产的单抗对PNGase F酶的酶解条件进行优化，包括缓冲液pH、酶种类、仪器、酶解程度、变性缓冲液及酶加入量等，总结酶解条件对N糖谱结果的影响。结果显示，置换缓冲液至1×PBS中可以避免某些单抗在低pH酶解时G0F转化为G0F-GN并可改善峰型；快速PNGase F和加入变性缓冲液能有效提高酶解效率；UPLC和1.7 μm粒径色谱柱能提高分离度；不完全酶解影响N糖含量结果。研究结果表明，采用优化的酶解条件可快速、有效的酶解单抗上的N糖，使N糖谱结果准确可靠，为细胞株筛选和单抗药物质量控制提供有效手段。     

A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.     

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.     

Analysis of N-linked oligosaccharides released from glycoproteins separated by two-dimensional gel electrophoresis

Protocols have been developed for the characterization of carbohydrate covalently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Mixtures of proteins (each at a level from 0.5 to 50 microg) were resolved in the first dimension according to their isoelectric points (pI), followed by separation in the orthogonal axis on the basis of their molecular weights. Glycans were released directly from excised gel spots after digestion with PNGase F, with or without prior treatment with trypsin. In a third method, glycoproteins were electroblotted onto poly(vinylidene difluoride) before glycans were released by PNGase F. For all these procedures profiles of the neutral and sialic acid-containing oligosaccharide mixtures were obtained after derivatization with 3-acetamido-6-aminoacridine, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or high-performance liquid chromatography. Potential applications to proteomics are discussed.     

Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS

This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and alpha1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.     

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.     

N-glycosylation of uterine endometrium determines its receptivity

Glycosylation alters the molecular and functional features of glycoproteins, which is closely related with many physiological processes and diseases. During “window of implantation”, uterine endometrium transforms into a receptive status to accept the embryo, thereby establishing successful embryo implantation. In this article, we aimed at investigating the role of N-glycosylation, a major modification type of glycoproteins, in the process of endometrial receptivity establishment. Results found that human uterine endometrial tissues at mid-secretory phase exhibited Lectin PHA-E+L (recognizes the branched N-glycans) positive N-glycans as measured by the Lectin fluorescent staining analysis. By utilizing in vitro implantation model, we found that de-N-glycosylation of human endometrial Ishikawa and RL95-2 cells by tunicamycin (inhibitor of N-glycosylation) and peptide-N-glycosidase F (PNGase F) impaired their receptive ability to human trophoblastic JAR cells. Meanwhile, N-glycosylation of integrin αvβ3 and leukemia inhibitory factor receptor (LIFR) are found to play key roles in regulating the ECM-dependent FAK/Paxillin and LIF-induced STAT3 signaling pathways, respectively, thus affecting the receptive potentials of endometrial cells. Furthermore, in vivo experiments and primary mouse endometrial cells-embryos coculture model further verified that N-glycosylation of mouse endometrial cells contributed to the successful implantation. Our results provide new evidence to show that N-glycosylation of uterine endometrium is essential for maintaining the receptive functions, which gives a better understanding of the glycobiology of implantation.     

Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.     

Glycomic alterations are associated with multidrug resistance in human leukemia

Correlations of disease phenotypes with glycosylation changes have been analyzed intensively in tumor biology field. In this study we describe glycomic alterations of multidrug resistance in human leukemia cell lines. Using multiple glycan profiling tools: real-time PCR for quantification of glycogenes, FITC-lectin binding for glycan profiling, and mass spectrometry for glycan composition, we compared the glycomics of drug-resistant K562/ADR cells with parental K562 line. The results showed that the expression of glycogenes, glycan profiling and N-glycan composition were different in K562/ADR cells, as compared with those in K562 cells, whereas O-glycans of the two cell lines showed no different mass spectra. Further analysis of the N-glycan regulation by way of tunicamycin application or PNGase F treatment in K562/ADR cells showed partial inhibition of biosynthesis and increased sensitivity to chemotherapeutic drugs in vitro. We targeted glycogene B3GNT8 and ST8SIA4, which were over-expressed in K562/ADR cells, and silenced the expression levels of two glycogenes after using RNA interference approach. The results showed that the silencing of B3GNT8 or ST8SIA4 in K562/ADR cells resulted in increased chemosensitivity to anti-tumor drugs. In conclusion, glycomic alterations are responsible for the overcoming multidrug resistance in human leukemia therapy and the N-linked oligosaccharides are associated with the drug resistance of cancer cells.     

Evidence of two enzymes performing the de-N-glycosylation of proteins in barley: expression during germination, localization within the grain and set-up during grain formation

The occurrence of two enzymes performing de-N-glycosylation of glycoproteins, namely, endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety). The dry grain showed both activities according to the HPLC detection of the hydrolysis of fluorescent resorufin-labelled substrates. However, PNGase activity was 16-fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5-fold compared to nearly 4.8-fold for ENGase over the 4 d following imbibition. The localization of these activities within the grain showed that the major contribution of PNGase was due to the endosperm, typically representing over 90% of the whole grain activity. In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10-fold higher than in the endosperm), particularly in the rootlets and scutellum. In developing spikes, PNGase activity was 5.6-fold higher than in the leaves, but similar ENGase activity was measured in both organs. During grain formation, PNGase activity followed dry matter increase together with endosperm development. In contrast, ENGase activity dropped by 66% at the beginning of grain filling before stabilizing until harvest. The occurrence of de-N-glycosylation-performing enzymes throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could represent the first evidence of specific functions for each enzyme.     

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.     

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.     

Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method

The mutant beta1,4-galactosyltransferase (beta4Gal-T1), beta4Gal-T1-Y289L, in contrast to wild-type beta4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, beta4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.