Structural features influencing hemagglutinin cleavability in a human influenza A virus.

The cleavability of the hemagglutinin (HA) molecule is related to the virulence of avian influenza A viruses, but its influence on human influenza virus strains is unknown. Two structural features are involved in the cleavage of avian influenza A virus HAs: a series of basic amino acids at the cleavage site and an oligosaccharide side chain in the near vicinity. The importance of these properties in the cleavability of a human influenza A virus (A/Aichi/2/68) HA was investigated by using mutants that contained or lacked an oligosaccharide side chain and had either four or six basic amino acids. All mutants except the one that contains a single mutation at the glycosylation site were cleaved, although not completely, demonstrating that a series of basic amino acids confers susceptibility to cellular cleavage enzymes among human influenza virus HAs. The mutants containing six basic amino acids at the cleavage site showed limited polykaryon formation upon exposure to low pH, indicating that cleavage was adequate to impart fusion activity to the HA. Deletion of the potential glycosylation site had no effect on the cleavability of these mutants; hence, the oligosaccharide side chain appears to have no role in human influenza virus HA cleavage. The inability to induce high cleavability in a human influenza A virus HA by insertion of a series of basic amino acids at the cleavage site indicates that other, as yet unidentified structural features are needed to enhance the susceptibility of these HAs to cellular proteases.     

寡糖氧化酶研究进展

寡糖氧化酶（oligosaccharide oxidase）是一种新型氧化酶,属于辅助活性家族7（auxiliary activity family 7,AA7）。其可作用的底物范围较广,能够催化多种寡糖氧化成相应的醛酸,并在反应过程中产生过氧化氢。根据寡糖氧化酶的作用底物,可将已报道的寡糖氧化酶分为葡糖寡糖氧化酶（gluco-oligosaccharide oxidase,GOOX）、木寡糖氧化酶（xylo-oligosaccharide oxidase,XOOX）和壳寡糖氧化酶（chito-oligosaccharide oxidase,COOX）等。寡糖氧化酶用途广泛,可应用于食品、医药、饲料、生物燃料等多种领域。但目前国内外对寡糖氧化酶的研究较少。基于此,从基本酶学性质、分子结构、作用机理、酶分子改良及应用等方面对寡糖氧化酶进行了综述,以期为寡糖氧化酶的实际研究及应用提供参考。     

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin.

The ability of many viruses to replicate in host cells depends on cleavage of certain viral glycoproteins, including hemagglutinin (HA). By generating site-specific mutant HAs of two highly virulent influenza viruses, we established that the relationship between carbohydrate in the stalk and the length of the connecting peptide is a critical determinant of cleavability. HAs that lacked an oligosaccharide side chain in the stalk were cleaved regardless of the number of basic amino acids at the cleavage site, whereas those with the oligosaccharide side chain resisted cleavage unless additional basic amino acids were inserted. This finding suggests that the oligosaccharide side chain interferes with HA cleavage if the number of basic amino acids at the cleavage site is not adequate to nullify this effect. Similar interplay could influence cleavage of other viral glycoproteins, such as those of human and simian immunodeficiency viruses and paramyxoviruses.     

壳寡糖对辣椒种子萌发及幼苗抗氧化酶活性影响研究

壳寡糖是甲壳素的重要衍生物,具有良好的生物学活性,可调节植物生长,使农作物和水果蔬菜增产丰收,因而在农业上的应用日渐增多,在农业上的应用,包括促进种子萌发和植物生长。本文选取辣椒种子为研究对象,探讨了不同浓度壳寡糖对辣椒种子萌发的影响,研究结果表明一定浓度的壳寡糖可以促进种子萌发,以0.10mg/L浓度的壳寡糖效果最显著;不同浓度壳寡糖浸种处理能激活辣椒幼苗抗氧化酶活性。     

Milk composition of a free-ranging African elephant (Loxodonta africana) cow during early lactation

Only one study previously reported comprehensively on the composition of African elephant's (Loxodonta africana) milk that was collected from 30 dead animals. In the current study milk was obtained from a tame but free-ranging African elephant cow without immobilization during the period when she was 4-47 days postpartum. At the respective collection times the nutrient content was 21.8 and 25.0 g protein; 56.0 and 76.0 g fat; 71.1 and 26.0 g sugars per kilogram of milk. The protein fraction, respectively, consisted of 10.0 and 14.0 g caseins/kg milk and of 11.8 and 11 g whey proteins/kg milk. During lactation the lactose content dropped from 52.5 to 11.8 g/kg milk, while the oligosaccharide content increased from 11.8 to 15.2 g/kg milk. The oligosaccharide was characterized as a galactosyllactose, which is digestible by cellulase. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in cow's milk, but some of the corresponding proteins were less negatively charged. The lipid fraction contains a high content of capric and lauric acids, approximately 60% of the total fatty acids, and low content of myristic, palmitic and oleic acids.     

Purification and characterization of GDP-L-fucose-N-acetyl beta-D-glucosaminide alpha 1----6fucosyltransferase from cultured human skin fibroblasts. Requirement of a specific biantennary oligosaccharide as substrate

GDP-L-fucose-N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase which catalyzes the transfer of fucose from GDP-L-fucose to the asparagine-linked N-acetyl-beta-D-glucosamine of N-linked glycoproteins has been purified 37,000-fold from cultured human skin fibroblasts. The Km values for the substrate asialoagalactotransferrin glycopeptide, and GDP-L-fucose were 66 and 4.2 microM, respectively. The Vmax was 1.4 mumols/mg/min. The key step in enzyme purification was affinity chromatography using the immobilized substrate asialoagalactotransferrin glycopeptide-CH-Sepharose. The affinity-purified enzyme had a minimum substrate requirement for a biantennary oligosaccharide with GlcNAc in terminal position, having a Km value of 55 microM. It was heretofore unexpected that the oligosaccharide would serve as substrate, since the site of enzyme activity is GlcNAc-1-linked to Asn. Although the presence of amino acids on this oligosaccharide enhanced the activity 3-fold, it is proposed that this may be the result of an alpha/beta anomeric mixture (2:1) of oligosaccharide used in these studies with only the beta anomer active as substrate. The implication is that the amino acid is required only to retain the beta anomeric position of the substrate. Removal of GlcNAc or addition of Gal to either the oligosaccharide or glycopeptide destroyed the ability to serve as substrates. In addition, di-N-acetylchitobiose, tri-N-acetylchitotriose and GlcNAc beta 1----Asn were nonpermissible substrates. This rigid substrate requirement is unique among fucosyltransferases thus far reported, since the natural substrates for the other enzymes may be substituted by one of several disaccharides.     

Lysosomal degradation of Asn-linked glycoproteins

Catabolism of Asn-linked glycoproteins to monosaccharides and amino acids occurs in lysosomes. Break-down must be complete to avoid lysosomal storage diseases that occur when fragments as small as dimers are left undigested. Recent results have clarified several aspects of Asn-linked glycoprotein catabolism in mammals. First, degradation of the oligosaccharide portion is accomplished by exo-glycosidases, which act only from the nonreducing end of chains to release sugar monomers as products. In contrast, proteolysis can proceed from both end and internal points along the polypeptide to eventually yield free amino acids. A second important feature of the glycoprotein disassembly pathway is that the hydrolytic steps can be grouped into two sets of ordered reactions: I) stepwise hydrolysis of the major portion of the oligosaccharide chains by a set of exoglycosidases, and II) ordered disassembly of the protein and the oligosaccharide-to-protein linkage region. Process II can vary at a single reaction step depending on the species in which degradation takes place. Thus, the last step of reaction sequence II can be either: 1) hydrolysis of the actual peptide-to-carbohydrate linkage, or 2) removal of the reducing-end GlcNAc from a previously freed oligosaccharide. The latter cleavage is catalyzed by the lysosomal glycosidase chitobiase. Chitobiase has been found only in humans and rats and not in other mammals (dogs, cats, goats, sheep, cats, or cattle). The hydrolytic mechanism of this enzyme is unique as it appears to be a reducing-end glycosidase and can be viewed as an accessory step in the human and rat digestive pathways. The species that lack this enzyme likely rely on exo-beta-D-glucosaminidase to cleave GlcNAc from both outer chain residues and the chitobiose moiety at the protein-to-carbohydrate linkage.     

N-Linked oligosaccharides of the H-2Dk histocompatibility protein heavy chain influence its transport and cellular distribution

The H-2K and H-2D proteins encoded by the K and D region of the major histocompatibility complex of the mouse were isolated by immunoprecipitation with specific antisera and resolved by two-dimensional gel electrophoresis. Of these two polypeptides, the H-2Dk glycoproteins isolated from macrophages of C3H/HeHa mice exhibit distinct cell surface and cytoplasmic forms although they share a strong degree of homology in the polypeptide backbone. Structurally they differ in their oligosaccharide structures. The structure of the oligosaccharides on the intracellular forms is of the high mannose type while the same structures on the cell surface forms are of the complex type. In the absence of all three oligosaccharide side chains, the unglycosylated polypeptides are expressed on the cell surface. In contrast, polypeptides containing one, two, or all three oligosaccharide side chains of the high mannose type are not transported to the cell surface. Cell surface expression of these glycoproteins requires processing of the oligosaccharide side chains from the high mannose form to the complex type. However, not all oligosaccharide antennae have to be terminally modified since H-2Dk glycoproteins synthesized in the presence of oligosaccharide-processing enzyme inhibitors such as swainsonine or monensin are also transported to the cell surface. H-2Dk glycoproteins containing oligosaccharide structures of the complex type but lacking terminal sialic acids are found on the cell surface, suggesting that sialylation is not required for transport. These results indicate that the oligosaccharide structures of the H-2Dk glycoproteins act to influence their cellular distribution.     

Tight binding of influenza virus hemagglutinin to its receptor interferes with fusion pore dilation

Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.     

Role of glycosylation in structure and stability of Erythrina corallodendron lectin (EcorL): a molecular dynamics study

The effect of glycosylation on structure and stability of glycoproteins has been a topic of considerable interest. In this work, we have investigated the solution conformation of the oligosaccharide and its effect on the structure and stability of the glycoprotein by carrying out a series of long Molecular dynamics (MD) simulations on glycosylated Erythrina corallodendron lectin (EcorL) and nonglycosylated recombinant Erythrina corallodendron lectin (rEcorL). Our results indicate that, despite the similarity in overall three dimensional structures, glycosylated EcorL has lesser nonpolar solvent accessible surface area compared to nonglycosylated EcorL. This might explain the experimental observation of higher thermodynamic stability for glycosylated EcorL compared to nonglycosylated EcorL. Analysis of the simulation results indicates that, dynamic view of interactions between protein residues and oligosaccharide is entirely different from the static picture seen in the crystal structure. The oligosaccharide moiety had dynamically stable interactions with Lys 55 and Tyr 53, both of which are separated in sequence from the site of glycosylation, Asn 17. It is possible that glycosylation helps in forming long-range contacts between amino acids, which are separated in sequence and thus provides a folding nucleus. Thus our simulations not only reveal the conformations sampled by the oligosaccharide, but also provide novel insights into possible molecular mechanisms by which glycosylation can help in folding of the glycoprotein by formation of folding nucleus involving specific contacts with the oligosaccharide moiety.     

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcβ1,4GlcNAcβ. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in β1,4-linkage to GlcNAc in vivo and in vitro. We now show that β4GalNAc-T3 and β4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to β1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galβ1,3(GalNAcβ1,4GlcNAcβ1,6)GalNAcαSer/Thr. The β1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcβ1,4GlcNAcβ (LacdiNAc)-containing structures on specific glycoproteins.     

An 18-kDa glycoprotein from bovine nasal cartilage. Isolation and primary structure of small, cartilage-derived glycoprotein

A glycosylated protein (small, cartilage-derived glycoprotein, SCGP) of approximately 18 kDa with unknown function has been isolated from dissociative extracts of bovine nasal cartilage and its primary structure determined. The protein has 121 amino acids, giving a calculated protein molecular weight of 13,878, four disulfide bonds, two N-linked oligosaccharides and one O-linked oligosaccharide. In nasal cartilage, this glycoprotein is in molar concentrations equivalent to 1/5-1/2 that of the link protein of cartilage proteoglycan aggregates, and it has also been isolated from bovine articular cartilage and from bovine fetal epiphysis. The N-terminal, glycosylated region of the molecule is relatively rich in arginine, proline, glycine, and threonine. The C-terminal 82 amino acids (which contains all four of the disulfide bonds and none of the carbohydrate) can be found as a discrete entity in cartilage extracts, indicating that the N-terminal domain is readily removed by extracellular proteolytic attack.     

Structure and significance of N-linked sugar unit of human serum amyloid P component

Human serum amyloid P component (SAP) was digested with pronase P and a glycopeptide fraction was obtained by gel-permeation chromatography. Carbohydrate and amino-acid composition of the glycopeptide suggested that each subunit of SAP possesses an N-linked glycan, but no O-linked ones. The N-linked oligosaccharide of SAP was obtained by hydrazynolysis. The structure of the oligosaccharide, which was deduced by sequential digestion with exoglycosidases and subsequent gel filtration, was identical or very similar to that of human transferrin. Removal of sialic acids from SAP reduced the calcium-dependent binding activity for agarose by 7%, suggesting the terminal sialic acids were partially responsible for the binding.     

Characterization of the Lipopolysaccharide from <Emphasis Type="Italic">Rahnella aquatilis</Emphasis> 1-95

The lipopolysaccharide from the freshwater bacterium Rahnella aquatilis 1-95 has been isolated and investigated for the first time. The structural components of the lipopolysaccharide molecule, such as lipid A, core oligosaccharide, and O-specific polysaccharide, were isolated by mild acidic hydrolysis. In lipid A, 3-hydroxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. In the core, oligosaccharide, galactose, arabinose, fucose, and an unidentified component were shown to be the major monosaccharides. The O-specific polysaccharide consists of a regularly repeating trisaccharide unit with the following structure: . Both acyl and phosphate groups have been shown to be responsible for the toxic and pyrogenic properties of the lipopolysaccharide of R. aquatilis.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 466–474.Original Russian Text Copyright © 2005 by Varbanets, E. Zdorovenko, Ostapchuk, G. Zdorovenko.     

Molecular modeling of glycosyltransferases involved in the biosynthesis of blood group A,blood group B,Forssman, and iGb3 antigens and their interaction with substrates

A terminal alpha1-3 linked Gal or GalNAc sugar residue is the common structure found in several oligosaccharide antigens, such as blood groups A and B, the xeno-antigen, the Forssman antigen, and the isogloboside 3 (iGb3) glycolipid. The enzymes involved in the addition of this residue display strong amino acid sequence similarities, suggesting a common fold. From a recently solved crystal structure of the bovine alpha3-galactosyltransferase complexed with UDP, homology modeling methods were used to build the four other enzymes of this family in their locked conformation. Nucleotide-sugars, the Mn2+ ion, and oligosaccharide acceptors were docked in the models. Nine different amino acid regions are involved in the substrate binding sites. After geometry optimization of the complexes and analysis of the predicted structures, the basis of the specificities can be rationalized. In the nucleotide-sugar binding site, the specificity between Gal or GalNAc transferase activity is due to the relative size of two clue amino acids. In the acceptor site, the presence of up to three tryptophan residues define the complexity of the oligosaccharide that can be specifically recognized. The modeling study helps in rationalizing the crystallographic data obtained in this family and provides insights on the basis of substrate and donor recognition.     

甘蓝与黄瓜寄主上B型烟粉虱和温室白粉虱蜜露糖分、氨基酸和挥发物组分的比较分析

粉虱蜜露是粉虱寄生性天敌搜索寄主的主要利它素源。应用离子色谱分别对甘蓝与黄瓜上B型烟粉虱(Bemisia tabaci B-biotype)蜜露以及黄瓜上温室白粉虱Trialeurodes vaporariorum蜜露的接触性利它素糖和氨基酸组分和含量进行了比较研究。结果表明：2种粉虱在不同寄主植物上的蜜露均富含糖和氨基酸,其中糖含量占绝对优势,甘蓝上B型烟粉虱蜜露、黄瓜上B型烟粉虱蜜露和黄瓜上温室白粉虱蜜露中的糖含量分别是相应氨基酸含量的42.5、2.6和5.4倍,其中糖类物质中又以寡糖含量占绝对优势,分别占89.3%、81.7%和88.2%。不同寄主植物和粉虱种类显著影响蜜露中糖和氨基酸的组成和含量。其中,甘蓝上B型烟粉虱蜜露中的寡糖以二糖占优势,占97.3%;二糖中又以蔗糖异构糖和松二糖占优势,分别占52.7%和35.4%。黄瓜上B型烟粉虱蜜露和温室白粉虱蜜露寡糖中以三糖和四糖占优势,分别占73.1%和85.4%;优势糖水苏（四）糖和松三糖分别占40.3%和 26.2%及49.9%和27.0%。甘蓝上B型烟粉虱蜜露中氨基酸以丙氨酸占优势,含量为66.5%;而黄瓜上B型烟粉虱及温室白粉虱蜜露中氨基酸以甘氨酸含量最高,分别占氨基酸总量的38.2%和51.7%。应用GC-MS对甘蓝上B型烟粉虱蜜露和黄瓜上温室白粉虱蜜露挥发物组分的鉴定结果显示,两种粉虱蜜露中共同含有的主要挥发物为邻苯二甲酸二（2-乙基）己酯。     

Characterization of the lipopolysaccharide from Rahnella aquatilis 1-95

The lipopolysaccharide from the freshwater bacterium Rahnella aquatilis 1-95 has been isolated and investigated for the first time. The structural components of the lipopolysaccharide molecule: lipid A, core oligosaccharide, and O-specific polysaccharide were isolated by mild acidic hydrolysis. In lipid A, 3-hydroxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. In the core oligosaccharide, galactose, arabinose, fucose, and an unidentified component were shown to be the major monosaccharides. The O-specific polysaccharide consists of a regularly repeating trisaccharide unit with the acyl and phosphate following structure: [structure: see text] groups have been shown to be responsible for the toxic and pyrogenic properties of the lipopolysaccharide of R. aquatilis.     

Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.