Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin

The glycoprotein IgM is the major antibody produced in the primary immune response to antigens, circulating in the serum both as a pentamer and a hexamer. Pentameric IgM has a single J chain, which is absent in the hexamer. The mu (heavy) chain of IgM has five N-linked glycosylation sites. Asn-171, Asn-332, and Asn-395 are occupied by complex glycans, whereas Asn-402 and Asn-563 are occupied by oligomannose glycans. The glycosylation of human polyclonal IgM from serum has been analyzed. IgM was found to contain 23.4% oligomannose glycans GlcNAc2Man5-9, consistent with 100% occupancy of Asn-402 and 17% occupancy of the variably occupied site at Asn-563. Mannan-binding lectin (MBL) is a member of the collectin family of proteins, which bind to oligomannose and GlcNAc-terminating structures. A commercial affinity chromatography resin containing immobilized MBL has been reported to be useful for partial purification of mouse and also human IgM. Human IgM glycoforms that bind to immobilized MBL were isolated; these accounted for only 20% of total serum IgM. Compared with total serum IgM, the MBL-binding glycoforms contained 97% more GlcNAc-terminating structures and 8% more oligomannose structures. A glycosylated model of pentameric IgM was constructed, and from this model, it became evident that IgM has two distinct faces, only one of which can bind to antigen, as the J chain projects from the non-antigen-binding face. Antigen-bound IgM does not bind to MBL, as the target glycans appear to become inaccessible once IgM has bound antigen. Antigen-bound IgM pentamers therefore do not activate complement via the lectin pathway, but MBL might have a role in the clearance of aggregated IgM.     

Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.     

Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin

Phaseolin, the major storage protein of the common bean (Phaseolus vulgaris), is a glycoprotein which is synthesized during seed development and accumulates in protein storage vacuoles or protein bodies. The protein has three different N-linked oligosaccharide side chains: Man9(GlcNAc)2, Man7(GlcNAc)2, and Xyl-Man3(GlcNAc)2 (where Xyl represents xylose). The structures of these glycans were determined by 1H NMR spectroscopy. The Man9(GlcNAc)2 glycan has the typical structure found in plant and animal glycoproteins. The structures of the two other glycans are shown below. (Formula; see text) Phaseolin was separated by electrophoresis on denaturing gels into four size classes of polypeptides. The two abundant ones have two oligosaccharides each, whereas the less abundant ones have only one oligosaccharide each. Polypeptides with two glycans have Man7(GlcNAc)2 attached to Asn252 and Man9(GlcNAc)2 attached to Asn341. Polypeptides with only one glycan have Xyl-Man3(GlcNAc)2 attached to Asn252. Both these asparagine residues are in canonical glycosylation sites; the numbering starts with the N-terminal methionine of the signal peptide of phaseolin. The presence of the Man7(GlcNAc)2 and of Xyl-Man3(GlcNAc)2 at the same asparagine residue (position 252) of different polypeptides seems to be controlled by the glycosylation status of Asn341. When Asp341 is unoccupied, the glycan at Asn252 is complex. When Asn341 is occupied, the glycan at Asn252 is only modified to the extent that 2 mannosyl residues are removed. The processing of the glycans, after the removal of the glucose residues, involves enzymes in the Golgi apparatus as well as in the protein bodies. Formation of the Xyl-Man3(GlcNAc)2 glycan is a multistep process that involves the Golgi apparatus-mediated removal of 6 mannose residues and the addition of 2 N-acetylglucosamine residues and 1 xylose. The terminal N-acetylglucosamine residues are later removed in the protein bodies. The conversion of Man9(GlcNAc)2 to Man7(GlcNAc)2 is a late processing event which occurs in the protein bodies. Experiments in which [3H]glucosamine-labeled phaseolin obtained from the endoplasmic reticulum (i.e. precursor phaseolin) is incubated with jack bean alpha-mannosidase show that the high mannose glycan on Asn252, but not the one on Asn341, is susceptible to enzyme degradation. Incubation of [3H] glucosamine-labeled phaseolin obtained from the Golgi apparatus with jack bean beta-N-acetylglucosaminidase results in the removal of the terminal N-acetylglucosamine residues from the complex chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-glycans of recombinant human interferon-gamma change during batch culture of chinese hamster ovary cells

A recombinant Chinese hamster ovary (CHO) cell line making human interfron-gamma (IFN-gamma) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-gamma was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alephl-6 fucosylated at Asn(25) but not at Asng(97)) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng(97) by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. (c) 1995 John Wiley & Sons, Inc.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Monoglucosylated glycans in the secreted human complement component C3: implications for protein biosynthesis and structure

The monoglucosylated oligomannose N-linked oligosaccharide (Glc(1)Man(9)GlcNAc(2)) is a retention signal for the calnexin-calreticulin quality control pathway in the endoplasmic reticulum. We report here the presence of such monoglucosylated N-glycans on the human complement serum glycoprotein C3. This finding represents the first report of monoglucosylated glycans on a human serum glycoprotein from non-diseased individuals. The presence of the glucose moiety in 5% of the human C3 glycoprotein suggests that this glycosylation site is sequestered within the protein and is consistent with previous studies identifying a cryptic conglutinin binding site on C3 that becomes exposed upon its conversion to iC3b.     

Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity

The properties of recombinant staphylokinase (SakSTAR) expressed in Pichia pastoris cells have been determined. The single consensus N-linked oligosaccharide linkage site in SakSTAR (at Asn28 of the mature protein) was occupied in approximately 50% of the expressed protein with high-mannose-type oligosaccharides. The majority of these glycans ranged in polymerization state from Man8GlcNAc2 to Man14GlcNAc2, with the predominant species being Man10GlcNAc2 and Man11GlcNAc2. Glycosylated SakSTAR (SakSTARg) did not differ from its aglycosyl form in its aggregation state in solution, its thermal denaturation properties, its ability to form a complex with human plasmin (hPm), the amidolytic properties of the respective SakSTAR-hPm complexes, or its ability to liberate the amino-terminal decapeptide required for formation of a functional SakSTAR-hPm plasminogen activator complex. However, this latter complex with SakSTARg showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same complex with the aglycosyl form of SakSTAR. We conclude that glycosylation at Asn28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but it is detrimental to the ability of the SakSTAR-hPm complex to serve as a hPg activator. This is likely due to restricted access of hPg to the active site of the SakSTARg-hPm complex.     

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.     

Interleukin-2 carbohydrate recognition modulates CTLL-2 cell proliferation

Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man(5)GlcNAc(2)Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor alpha, beta, gamma subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor alpha subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-beta-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man(5)GlcNAc(2) is linked to the extracellular portion of the IL-2 receptor alpha subunit. Our findings indicate that IL-2 binds the IL-2 receptor alpha subunit through Man(5)GlcNAc(2) and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Ralpha subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Ralpha, -beta, and -gamma subunits, leading to cellular signaling.     

Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins

The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.     

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.     

Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.     

The effect of the protein matrix on glycoprotein processing by oviduct Golgi enzymes

Using the avidin-biotinyl glycan system reported previously (Shao, M.-C., and Wold, F. (1987) J. Biol. Chem. 267, 2968-2972), we have compared the processing efficiency of oviduct enzymes acting on different glycan-(biotinyl)Asn and glycan-(6-biotinamidohexanoyl)Asn derivatives when they are free and bound to avidin. The glycans were selected to permit exploration of the individual processing steps, and the two different groups of derivatives were used to assess both the close (biotinyl) and more distal (biotinamidohexanoyl) display of the glycan relative to the avidin surface. The direct comparison of the free and avidin-bound glycans demonstrated that mannosidase I is strongly inhibited by avidin in both the close and distal complexes, whereas GlcNAc transferase I and mannosidase II are strongly inhibited only in the close complex. GlcNAc transferases III, IV, and V, which could only be assessed individually by indirect means using different substrates, did not appear to be affected in any major way by the protein matrix; the data suggest that transferase III is inhibited only to a minor extent in the close complex. Gal transferase activity showed a minor effect of the avidin matrix for both complexes in the hybrid processing pathways. The most significant consequence of the avidin effect on Gal transferase was the apparent abolishment of the incorporation of a 2nd Gal residue in the two avidin complexes. This survey of the protein matrix effects on glycan processing by oviduct enzymes appears to provide reasonable clues to the origin of the very different glycan structures observed in oviduct-processed glycoproteins. Thus, ovalbumin and avidin itself, containing a mixture of oligomannose and hybrid glycans at their single glycosylation sites, may well present they glycans to the processing enzymes in a display very similar to that of the avidin close complex observed here. The inhibition of mannosidase I and GlcNAc transferase I lead to preservation of oligomannose structures, whereas the strong inhibition of mannosidase II favors the incorporation of the bisecting GlcNAc by GlcNAc transferase III to yield hybrid structures as the most processed products. Ovomucoid, which contains multiantennary complex structures at all glycosylation sites, may on the other hand display its glycans, unencumbered by the protein surface, in conformations similar to either the free glycans or the distal complexes observed in this work.     

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.     

Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module.

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.     

Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus

Glycosylation is of importance for the structure and function of proteins. In the case of vitellin (Vt), a ubiquitous protein accumulated into granules as the main yolk protein constituent of oocytes during oogenesis, glycosylation could be of importantance for the folding, processing and transport of the protein to the yolk and also provides a source of carbohydrate during embryogenesis. Vt from the crayfish Cherax quadricarinatus is synthesized as a precursor protein, vitellogenin (Vg), in the hepatopancreas, transferred to the hemolymph, and mobilized into the growing oocyte via receptor-mediated endocytosis. The gene sequence of C. quadricarinatus shows a 2584-amino-acid protein with 10 putative glycosylation sites. In this study a combined approach of lectin immunoblotting, in-gel deglycosylation, and mass spectrometry was used to identify the glycosylation sites and probe the structure of the glycan moieties using C. quadricarinatus Vg as a model system. Three of the consensus sites for N-glycosylation-namely, Asn(152), Asn(160) and Asn(2493)-were glycosylated with the high-mannose glycans, Man(5-9)GlcNAc(2), and the glucose-capped oligosaccharide Glc(1)Man(9)GlcNAc(2).     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Glycosylation of interleukin-6 purified from normal human blood mononuclear cells.

Interleukin 6 (IL-6) is a glycosylated cytokine which is important in exerting cell-specific growth-inducing, growth-inhibiting and differentiation-inducing effects. IL-6 produced in mammalian cell lines is heterogeneous, reflecting specific cell-type-dependent post-translational modifications. Native IL-6 was purified from human blood mononuclear cells and the oligosaccharides released, radiolabelled and sequenced by a combination of sequential exoglycosidase digestion using Bio-Gel P-4 high-resolution gel chromatography and acetolysis. N- and O-linked glycans were found. The N-linked glycans were sialylated di- and tri-antennary complex-type and oligomannose-type structures. However, the most predominant N-linked oligosaccharide was a small tetrasaccharide with the sequence Man alpha 6Man beta 4GlcNAc beta 4GlcNAc. This is the first report of this structure on a circulating glycoprotein. This structure has only previously been reported to be present on the syncytiotrophoblast of human placenta. The presence of the oligomannose structures and the mannose-terminating tetrasaccharide on IL-6 may be important in maintaining a high local concentration of the cytokine while limiting its systemic serum level via interaction with soluble mannose-binding serum lectins.     

Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale.

The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine-histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is approximately 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of approximately 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1-->6(Man1-->3) (Xyl1-->2)Man1-->4GlcNAc1-->4(Fuc1-->3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.     

Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.