Biochemical Studies on “Bakanae” Fungus. Part 68. Synthesis of Substances related to Gibberellins

Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-β-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-β-N-acetylglucosaminidases of other microorganisms: ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins.     

The expression of free oligosaccharides in human seminal plasma

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.     

研究: 奶牛与水牛初乳中乳寡糖组分比较研究

乳寡糖是由乳汁中含量丰富的固体物质组成．研究结果表明,乳寡糖有提高免疫、益生元及抗感染等作用,已发现与婴儿肠道发育、神经智力发育等多方面关系密切．水牛奶是除牛奶外的第二大奶源,国际上公认其为营养含量高、口感好的优质乳制品,但目前针对水牛乳寡糖的研究多以美洲水牛为研究对象,尚无中国水牛的相关研究．本研究利用固相萃取对已脱脂和除去蛋白质的广西水牛初乳乳汁样品进行纯化,并采用苯胺 (aniline,Bn)衍生化试剂对其进行衍生化处理,通过UPLC-ESI-Q-TOF-MS液相质谱进行优化后,对水牛初乳中的寡糖组分进行测定并与牛乳进行了对比,最终测得奶牛初乳中19种及水牛初乳中的9种乳寡糖组分,并对二者的种类及含量进行比较,发现在两种初乳的乳寡糖中,中性糖二糖m/z 385.15和中性糖三糖m/z 547.21以及酸性糖m/z 635.23均为其主要寡糖成分,与其他乳寡糖相比含量相对较高．总体而言水牛初乳中的中性寡糖占比比奶牛初乳高,二者中性糖占乳寡糖总量的比例分别为88.88%和63.16%．     

Novel High-Molecular Weight Fucosylated Milk Oligosaccharides Identified in Dairy Streams

Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called “mother liquor”, and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk.     

Structures of the asparagine-linked sugar chains of laminin

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.     

Evolution of milk oligosaccharides: Origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals

BackgroundThe carbohydrate fraction of mammalian milk is constituted of lactose and oligosaccharides, most of which contain a lactose unit at their reducing ends. Although lactose is the predominant saccharide in the milk of most eutherians, oligosaccharides significantly predominate over lactose in the milk of monotremes and marsupials.Scope of reviewThis review describes the most likely process by which lactose and milk oligosaccharides were acquired during the evolution of mammals and the mechanisms by which these saccharides are digested and absorbed by the suckling neonates.Major conclusionsDuring the evolution of mammals, c-type lysozyme evolved to α-lactalbumin. This permitted the biosynthesis of lactose by modulating the substrate specificity of β4galactosyltransferase 1, thus enabling the concomitant biosynthesis of milk oligosaccharides through the activities of several glycosyltransferases using lactose as an acceptor. In most eutherian mammals the digestion of lactose to glucose and galactose is achieved through the action of intestinal lactase (β-galactosidase), which is located within the small intestinal brush border. This enzyme, however, is absent in neonatal monotremes and macropod marsupials. It has therefore been proposed that in these species the absorption of milk oligosaccharides is achieved by pinocytosis or endocytosis, after which digestion occurs through the actions of several lysosomal acid glycosidases. This process would enable the milk oligosaccharides of monotremes and marsupials to be utilized as a significant energy source for the suckling neonates.General significanceThe evolution and significance of milk oligosaccharides is discussed in relation to the evolution of mammals.     

Endo β-N-acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides

Endo β-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N′ -diacetylchitobiose, N,N′ -diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase)mixture readily leaved high mannose and complex oligosaccharides (neutral and sialyated) with common core α1–6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core α1–6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides. However, it was similar to Endo H when reduced ovalbumin oligosaccharides were used as substrates, consistent with the recently isolated Endo F subfraction F₁ being similar to Endo H [Trimble, R. B. and Tarentino, a. L. (1991). J. Biol. Chem. 266, 1646]. Results obtained in this study suggest that the complex oligosaccharides cleaving enzymes F₂ and F₃ show high specificity towards peptide free oligosaccharides with the core α1-6 linked fucose, unlike the glycopeptide substrates. Therefore PNGase free Endo F₁, F₂ and F₃ mixtures should be useful in the functional evaluation of the oligosaccharides in glycoproteins.     

Lignocellulose derived functional oligosaccharides: production,properties, and health benefits

Abstract

Lignocellulosic biomass (LB) is the renewable feedstock for the production of fuel/energy, feed/food, chemicals, and materials. LB could also be the versatile source of the functional oligosaccharides, which are non-digestible food ingredients having numerous applications in food, cosmetics, pharmaceutical industries, and others. The burgeoning functional food demand is expected to be more than US$440 billion in 2022. Because of higher stability at low pH and high temperature, oligosaccharides stimulate the growth of prebiotic bifidobacteria and lactic acid bacteria. Xylooligosaccharides (XOS) are major constituents of oligosaccharides consisting of 2–7 xylose monomeric units linked via β-(1,4)-linkages. XOS can be obtained from various agro-residues by thermochemical pretreatment, enzymatic or chemoenzymatic methods. While thermochemical methods are fast, reproducible, enzymatic methods are substrate specific, costly, and produce minimum side products. Enzymatic methods are preferred for the production of food grade and pharmaceutically important oligosaccharides. XOS are potent prebiotics having antioxidant properties and enhance the bio-adsorption of calcium and improving bowel functions, etc. LB can cater to the increasing demand of oligosaccharides because of their foreseeable amount and the advancements in technology to recover oligosaccharides. This paper summarizes the methods for oligosaccharides production from LB, classification, and benefits of oligosaccharides on human health.     

Production of mannose-containing oligosaccharides by glucansucrase E81 and determination of their functional characteristics

Abstract

Oligosaccharides are one of the functional ingredients to be used in food technology. In this study, by using an active glucansucrase GTFA-ΔN E81 in the acceptor reaction of mannose, mannose-containing oligosaccharides were produced and their functionality was tested. The formation of the oligosaccharides were visualised by TLC analysis and mannose-containing oligosaccharides up to DP 7 were determined by LC-MS analysis. The presence of the (1,6)Glc and (1,3)Glc units within the oligosaccharides were determined by NMR analysis. The in vitro immune-modulatory functions of the mannose-containing oligosaccharides were determined but no induction in IL-4, IL-10, IL-12 and TNF-α cytokine levels were detected. Importantly this oligosaccharide mixture showed prebiotic effect by triggering the growth of tested probiotics and not affecting the growth of pathogen strains. Our findings reveals the potential of the role of glucansucrases for the production of functional oligosaccharides.     

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.     

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of α1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of α1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K_d) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for α1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K_a) for α1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were α1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of α1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.     

Oligosaccharide alterations of rat glioblast membrane-bound glycoproteins during differentiation induced by glia maturation factor

Differentiation of normal glioblasts was induced by glia maturation factor (GMF), and the structural change in the oligosaccharide chains of the plasmalemmal glycoproteins was investigated. After the glycopeptides obtained by trypsin treatment of the intact cells had been digested with pronase, the resulting glycopeptides were separated into 4 fractions by gel filtration. The first 2 fractions were found to contain mainly N-glycosidically linked glycopeptides, and the last 2, O-linked oligosaccharides. There were a variety of N-linked oligosaccharides whose apparent molecular weights were greater than that of isomaltoheptaose. As compared to those, O-linked oligosaccharides were fewer in type and lower in molecular weight. The N-linked oligosaccharides corresponding to isomaltohepta- decaose and larger saccharide chains augmented in differentiated glioblasts, whereas the N-linked oligosaccharides smaller than isomaltoheptade- caose decreased. The turnover rate of the high molecular weight oligosaccharides was faster than that of other membrane oligosaccharides, and was accelerated by GMF treatment. The content of an O-linked oligosaccharide fraction increased after GMF treatment.     

Specific adsorption of starch oligosaccharides in the gel phase of starch granules

The gel phase of native starch-granules is penetrable by such low-molecular-weight solutes as oligosaccharides, amino acids, and salts [Lathe and Ruthven, Biochem. J., 62 (1956) 665]. Molecules larger than about 1000 daltons are effectively excluded. Starch oligosaccharides (maltotriose through maltoheptaose and perhaps higher) exhibit anomalous behavior in that they are taken up by the gel phase far in excess of the amount expected on the basis of their molecular size. Adsorption was measured by using radioactive starch oligosaccharides and counting weighed amounts of solution before and after equilibration with starch granules. The measurements were corrected for water sorption by the starch granules and for exclusion effects as ascertained by controls with nonstarch types of oligosaccharides. Maximum adsorption was observed with maltotetraose. The results indicate a specific binding between the starch oligosaccharides and molecular chains in the starch, presumably those chains in the gel phase. We suggest that these chains constitute interbranch regions of branched molecules, or segments of linear molecules in the gel or amorphous phase, the segments being of sufficient length to form a double helix or other association with the linear oligosaccharides.     

Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells

The glycosylation pattern of a humanized anti-EGFR×anti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody.     

Structures of the oligosaccharides isolated from milk of the platypus

Eight major oligosaccharides were isolated from platypus milk. By sequential exoglycosidase digestion and methylation study, their structures were elucidated as shown in Fig. 9 of this paper. The characteristics feature of the platypus milk oligosaccharides is that lacto-N-neotetraose and lacto-N-neohexaose are the major cores in contrast to human milk oligosaccharides in which lacto-N-tetraose and lacto-N-hexaose are found as the major core.     

Solvolytic depolymerization of chondroitin and dermatan sulfates

It is essential to establish a library of glycosaminoglycan oligosaccharides from the chondroitin and dermatan sulfates to investigate their biological functions and structure-activity relationships (SARs). There are several approaches to obtain oligosaccharides using chemical and enzymatic degradation procedures; however, purification of each resulting oligosaccharide is complicated because of the diversity of sulfonation patterns present in these oligosaccharides. We have developed a new method for the solvolytic degradation for chondroitin and dermatan sulfates to obtain an oligosaccharide mixture that can be easily purified into chondro/dermato oligosaccharides for characterization by both ¹H NMR and MALDI-TOFMS. These oligosaccharides have a methyl-esterified uronate residue and a methyl 2-acetamido-2-deoxy-d-galactofuranoside at the nonreducing and reducing ends, respectively. All other internal repeating disaccharide units were desulfonated, but maintained their core carbohydrate structures.     

Bifidogenic effect and in vitro immunomodulatory roles of melibiose-derived oligosaccharides produced by the acceptor reaction of glucansucrase E81

Glucansucrases are responsible for the production of α-glucans using sucrose as the substrate. Glucansucrases may also produce different oligosaccharides by transferring the glucose moiety from sucrose to a variety carbohydrates acting as acceptor nucleophile. In this study, melibiose-derived oligosaccharides were produced by the glucansucrase from Lactobacillus reuteri E81 expressed without the N-terminal region (GtfA-ΔN). The reaction products were characterized by TLC, LC-MS and NMR analysis and it was found that GtfA-ΔN synthesized melibiose-derived oligosaccharides (DP3 and DP4) by adding glucose units through alpha 1->3 or 1->6 glycosidic bond. The functional characteristics of these melibiose-derived oligosaccharides were determined by testing the immune-modulatory functions in HT-29 cells and testing their growth promoting effects for important probiotic and pathogenic strains. The melibiose-derived oligosaccharides triggered the production of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine TNF-α depending on their concentrations. Finally, melibiose-derived oligosaccharides showed bifidogenic effect as potential prebiotics.     

On-line cleanup for 2-aminobenzamide-labeled oligosaccharides

Analysis of 2-aminobenzamide-labeled oligosaccharides requires removal of excess labeling reagents before chromatography. Manual cleanup is time-consuming and not optimal for routine analysis, so an on-line solid-phase extraction was developed. Labeled oligosaccharides are trapped on an amide phase in a small guard column, the excess reagents are washed away, and then the sample is transferred to the analytical column for analysis. The on-line protocol shortened the sample preparation time and has been applied for the analysis of oligosaccharides and N-glycans released from glycoproteins.     

Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica.

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242] to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor. The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by high-resolution proton NMR. Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N-acetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal alpha-galactose residue(s) linked to C3 of beta-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Gal beta 1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.     

Heparin oligosaccharides enhance tissue-type plasminogen activator: a correlation between oligosaccharide length and stimulation of plasminogen activation

The rate of plasminogen (Pg) activation by tissue-type Pg activator (t-PA) is enhanced by heparin-derived oligosaccharides. Kinetic analysis of the effects of heparin oligosaccharides, ranging in size from di- to dodecasaccharides, on Pg activation demonstrates that stimulation of the reaction is dependent on the size of the heparin oligosaccharides. Di- and tetrasaccharides enhance the activation through 2-fold increases in kcat and 4-fold decreases in Km. Hexasaccharide and larger oligosaccharides stimulate the reaction by increasing the kcat by as much as 4-fold, but do not affect the Km. Previous experiments have shown that lipoprotein(a) [Lp(a)] inhibits Pg activation by t-PA, but only in the presence of a template which enhances t-PA activity such as fibrinogen fragments or intact heparin. Similiarly, Lp(a) inhibits the enhancement of t-PA activity by the larger heparin oligosaccharides but has no effect on t-PA activity in the presence of di- and tetrasaccharides. The results of this study when considered with our previous observations (Edelberg & Pizzo, 1990) suggest that the enhancement in Pg activation by the smaller oligosaccharides is mediated exclusively via binding to t-PA while the larger oligosaccharides may interact with both t-PA and Pg. Furthermore, studies of Pg activation in the presence of both heparin oligosaccharides and fibrinogen fragments demonstrate that t-PA is stimulated preferentially by fibrinogen fragments.