Synthesis of glycosylated peptide templates containing 6′-O-phosphorylated mannose disaccharides and their binding to the cation-independent mannose 6-phosphate receptor

Christensen, Mette K.; Meldal, Morten; Bock, Klaus; Cordes, Henriette; Mourit­sen, Søren; Elsner, Henrik I.

doi:10.1039/p19940001299

Cited by 47 publications

(33 citation statements)

References 30 publications

(8 reference statements)

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“…First, the effect of a small bivalent Man-6-P-containing peptide on the rate of IGF-II uptake was determined. The peptide, a Thr-Lys-Thr tripeptide with a Man-6-P(␣1-2)Man disaccharide attached to each threonine, has an affinity for the M6P/IGF-II receptor that is similar to that of an oligosaccharide with two Man-6-P residues and over 1000-fold higher than that of Man-6-P (23,30,31). This high binding affinity indicates that the ligand is interacting with two binding sites on the M6P/IGF-II receptor.…”

Section: Intermolecular Cross-linking Of M6p/igf-ii Receptors Is Respmentioning

confidence: 99%

The Rate of Internalization of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is Enhanced by Multivalent Ligand Binding

York

Arneson

Gregory

et al. 1999

Journal of Biological Chemistry

105

117

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The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, ␤-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that ␤-glucuronidase entered the cell ϳ3-4-fold faster than IGF-II. Unlabeled ␤-glucuronidase stimulated the rate of internalization of 125 I-IGF-II to equal that of 125 I-␤-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize ␤-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with ␤-glucuronidase generated a complex composed of two receptors and one ␤-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) 1 is a type I transmembrane glycoprotein that cycles through the Golgi, endosomes, and the plasma membrane to carry out its role in the biogenesis of lysosomes and in the clearance of the polypeptide insulin-like growth factor II (IGF-II) (1, 2). In the Golgi, the receptor binds newly synthesized acid hydrolases modified with mannose 6-phosphate (Man-6-P) residues on their asparagine-linked oligosaccharides and transports them to endosomes via clathrincoated vesicles (3-5). The acid hydrolases are released in the acidified endosome and then packaged into lysosomes while the receptor either returns to the Golgi to bind another ligand or moves to the plasma membrane (6, 7). At the plasma membrane, the M6P/IGF-II receptor mediates internalization of Man-6-P-containing ligands and IGF-II (3,5,8).The interactions of IGF-II and Man-6-P-containing ligands with the M6P/IGF-II receptor have been characterized in several studies (8 -12). The extracellular portion of the M6P/ IGF-II receptor contains 15 homologous repeating domains of ϳ147 amino acids each (13). Domains 3 and 9 (numbering from the amino terminus) each bind 1 mol of Man-6-P, and the single IGF-II-binding site has been mapped to domain 11 in the extracellular region (14 -16). Man-6-P residues do not inhibit binding of IGF-II to the receptor, verifying that the two ligandbinding sites are distinct. However, proteins containing Man-6...

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Section: Intermolecular Cross-linking Of M6p/igf-ii Receptors Is Respmentioning

confidence: 99%

The Rate of Internalization of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is Enhanced by Multivalent Ligand Binding

York

Arneson

Gregory

et al. 1999

Journal of Biological Chemistry

105

117

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“…NN-Dimethylformamide (DMF) was distilled. Tyr(NO2), 2-aminobenzoic acid (Fluka), and FmocLys(Boc)-OH were transformed into Fmoc-Tyr(NO2)-OH, Boc-ABz-ODhbt, and Fmoc-Lys(BocABz)-OPfp, respectively, as described (5,32), where Fmoc is fluoren-9-ylmethyloxycarbonyl, Boc is tert-butyloxycarbonyl, Dhbt is 3,4-dihydro-4-oxo-1,2,3-benzotriazo-3-yl, and Pfp is pentafluorophenyl. Fmoc-amino acid-OPfp esters were from MilliGen or Bachem.…”

Section: Methodsmentioning

confidence: 99%

Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

Meldal

Svendsen

Breddam

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

179

145

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A solid-phase assay for the complete subsite mapping doftheactive site ofeldop eaes has been developed.A lbrary of resin-bound ptase ubstrates was sytsed both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(NN-dlnethylacryhmlde) copolymer type of resin that alows proteas to diffuse into the interior and perform their catalytic activity. An lic acid and 3-it rosine were used a an efclet donor-acceptor p for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet ming of the bead and ll washing p eus. After treatment wit subtiAsin Carlsberg, beads were c and subjected to peptide seq , affording the preferred s quences, their cleavage bond, and a esiqstimation of the turnover. A satitcal dbution of prefed amino acids was obtain for each subsite. The result was compaed with data from kinetic studies In solution.In early studies of the activity of isolated proteolytic enzymes, polypeptides were subjected to digestion with the enzyme (1) and the preferred cleavage site, if any, was determined by isolation of the fragments and subsequent Edman degradation. This often tedious work provided lead amino acid sequences that could be used for determination of the optimal substrates. These were prepared by chemical synthesis and the kinetic constants were measured by determination ofthe rate ofproduct formation by HPLC (2), NMR spectroscopy (3), or spectrophotometric monitoring when chromogenic or fluorogenic substrates could be used (4, 5). More recently the development of internally quenched fluorogenic substrates of the resonance energy transfer type (6, 7) has facilitated the complete subsite mapping (5,(8)(9)(10)(11)(12) for endoproteases. In these substrates, it is important that efficient long-range energy transfer is observed between the donor and the acceptor to span the entire active site of the endopeptidase, thus minimizing the interaction between the enzyme and the chromophoric probes. The donor-acceptor amino acid pair o-aminobenzamide (ABz)-3-nitrotyrosine [Tyr(NO2)] (13) for which excellent quenching of fluorescence is observed has recently been described (5). This donor-acceptor pair is conveniently introduced in parallel multiple-column peptide synthesis (MCPS) (14) of numerous substrates and has been used for subsite mapping of a variety of proteases (6,(15)(16)(17)(18)(19)(20)(21). However, substantial effort is still required to identify the optimal substrates.The use ofcombinatorial peptide libraries (22) and portionmixing libraries (23, 24) is widely accepted as the method of choice for defining binding motifs and unknown biological activities. The portion-mixing library is particularly convenient for the presentation of millions of substrates to a protease with unknown specificity. The problem is, however, to detect, isolate, and characterize the active substances. Due to the high quantum yield of the ABz group, the fluorescence can easily be observed visually in the absence of Tyr(N02), whereas peptides containi...

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“…Glycopeptides may be interesting targets to modulate galectin binding because the carbohydrate part provides the specificity of the interaction, whereas the peptide backbone may actively participate in binding by, for example, hydrogen bonding or hydrophobic interactions. Glycopeptides can be generated in a library format via a combinatorial approach [7,8]. The most frequently implemented method for the generation of ''one-bead-one-compound" (glyco)peptide libraries [9,10] is the split-and-mix method [11,12].…”

mentioning

confidence: 99%

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

Maljaars¹,

André²,

Halkes³

et al. 2008

Analytical Biochemistry

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a b s t r a c tCombinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(b1-O)Thr, Gal(b1-S)Cys/Gal(b1-N)Asn, and Lac(b1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.

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Synthesis of glycosylated peptide templates containing 6′-O-phosphorylated mannose disaccharides and their binding to the cation-independent mannose 6-phosphate receptor

Cited by 47 publications

References 30 publications

The Rate of Internalization of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is Enhanced by Multivalent Ligand Binding

The Rate of Internalization of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is Enhanced by Multivalent Ligand Binding

Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

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