N-Glycan Modification in Aspergillus Species

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man₅GlcNAc₂ type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan₅GlcNac₂ N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man₃GlcNAc₂ N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.     

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1–TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.     

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of α-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.     

Glycosylation of site-specific glycans of α1-acid glycoprotein and alterations in acute and chronic inflammation

Backgroundα₁-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation.MethodsAGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.ResultsSite-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in α1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary α1,3-fucosylation at sites 3 and 4 and tetra-antennary α1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of α1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4.ConclusionLittle variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and α1,3-fucosylated N-glycan structures at each glycosylation site.     

Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis

Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.     

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana

Background

The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.

Methodology/Principal Findings

Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.

Conclusions/Significance

We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.     

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

N-glycans modulate the function of human corticosteroid-binding globulin

Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5-99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0-34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues.     

Single-cell proteomic analysis of glucosinolate-rich S-cells in Arabidopsis thaliana

Single-cell analysis is essential for understanding the processes of cell differentiation and metabolic specialisation in rare cell types. The amount of single proteins in single cells can be as low as one copy per cell and is for most proteins in the attomole range or below; usually considered as insufficient for proteomic analysis. The development of modern mass spectrometers possessing increased sensitivity and mass accuracy in combination with nano-LC–MS/MS now enables the analysis of single-cell contents. In Arabidopsis thaliana, we have successfully identified nine unique proteins in a single-cell sample and 56 proteins from a pool of 15 single-cell samples from glucosinolate-rich S-cells by nanoLC–MS/MS proteomic analysis, thus establishing the proof-of-concept for true single-cell proteomic analysis. Dehydrin (ERD14_ARATH), two myrosinases (BGL37_ARATH and BGL38_ARATH), annexin (ANXD1_ARATH), vegetative storage proteins (VSP1_ARATH and VSP2_ARATH) and four proteins belonging to the S-adenosyl-l-methionine cycle (METE_ARATH, SAHH1_ARATH, METK4_ARATH and METK1/3_ARATH) with associated adenosine kinase (ADK1_ARATH), were amongst the proteins identified in these single-S-cell samples. Comparison of the functional groups of proteins identified in S-cells with epidermal/cortical cells and whole tissue provided a unique insight into the metabolism of S-cells. We conclude that S-cells are metabolically active and contain the machinery for de novo biosynthesis of methionine, a precursor for the most abundant glucosinolate glucoraphanine in these cells. Moreover, since abundant TGG2 and TGG1 peptides were consistently found in single-S-cell samples, previously shown to have high amounts of glucosinolates, we suggest that both myrosinases and glucosinolates can be localised in the same cells, but in separate subcellular compartments. The complex membrane structure of S-cells was reflected by the presence of a number of proteins involved in membrane maintenance and cellular organisation.     

Characterizing the Link between Glycosylation State and Enzymatic Activity of the Endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana

Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.     

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites (NGS). Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species: Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Our analysis shows that 78% of all asparagines of NXS/T motif involved in N-glycosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence (S/TXN) shows a similar distribution across the secondary structural elements, indicating that the NXS/T motif in itself is not biologically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool (SFAT), is freely available to the public at http://hive.biochemistry.gwu.edu/tools/sfat.     

N-glycosylation sites of plant purple acid phosphatases important for protein expression and secretion in insect cells

Analysis of plant purple acid phosphatases (PAPs) showed high conservation and different distribution of N-glycosylation sites. Oligosaccharide structures of Lupinus luteus acid phosphatase (Lu_AP) produced in insect cells were determined. Mutant Lu_AP and Phaseolus vulgaris (Ph_AP) phosphatases lacking possibility of N-glycosylation at highly conserved sites were generated and expressed in insect cells. A role for N-glycosylation in the stability of PAPs was indicated by unsuccessful attempts to secrete Ph_AP and Lu_AP mutants generated by replacing Asn residues of conserved glycosylation sequons by Ser residues either singly or in combination. We showed that Ph_AP belongs to the group of glycoproteins that require occupancy of all highly conserved glycosylation sites for secretion, whereas replacing of the third position of the glycosylation sequon indicated that Lu_AP may tolerate the absence of some N-glycans. However, the N-glycan located at the polypeptide C-terminus was crucial for secretion of both enzymes. PAP specific activity of glycosylation mutants successfully secreted was similar to the wild-type recombinant proteins.     

Isolation of glycopeptides containing individual glycosylation sites of Friend murine leukemia virus glycoprotein: studies of glycosylation by methylation analysis

Glycopeptides containing individual N-glycosylation sites of the glycoprotein from Friend murine leukemia virus were isolated by digestion of the viral glycoprotein with protease of S. aureus (V8) or with trypsin followed by fractionation of the resulting (glyco)peptides by gel filtration and reversed-phase, high-performance liquid chromatography at pH 6. Isolated glycopeptides were assigned to the known amino acid sequence of the protein by amino acid analysis and by determination of the NH2-termini. The carbohydrate moieties of each glycosylation site were analysed by methylation analysis. A high selectivity of the glycoprotein glycosylation was found with regard to the distribution of oligomannosidic, mixed, and N-acetyl-lactosaminic oligosaccharides.     

Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non–small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.     

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.