Synthetic glycopeptide substrates for receptor-mediated endocytosis by macrophages.

Abstract

Mammalian macrophages contain a transport system that binds and internalizes glycoproteins with exposed mannose residues. This system and analogous systems on other types of cells require substrates to bear multiple nonreducing terminal residues ofthe appropriate sugar for effective uptake. Small multivalent synthetic glycopeptides with mannose residues covalently linked through a spacer arm to the a-and e-amino groups of lysine, dilysine, and trilysine are competitive inhibitors of rat alveolar macrophage uptake of the neoglycoprotein mannosyl-bovine serum albumin with inhibition constants in the ,uM range. Various compounds could be covalently attached to the a-carboxyl group of these glycopeptides with substantial retention of#hibi-tory potency. This uptake system does not recognize galactose residues, and the galactosyl analog of an inhibitory mannosylpeptide did not inhibit uptake of mannosyl-bovine serum albumin. The trimannosyldilysine ligand is not only an inhibitor but also a substrate for specific uptake by macrophages, as shown with an 125F labeled derivative. Macrophages bound 6.4 x 105 molecules per cell at 0°C with a dissociation constant of 2 ,uM. At 21°C the cells could internalize the labeled conjugate with an apparent Michaelis constant of 6 ,uM and a maximal velocity of 1.7 x 105 molecules per min per cell. The dissociation constant and Michaelis constant are similar to the inhibition constant of 9 ,uM determined at 21°C for inhibition by this conjugate of mannosyl-bovine serum albumin uptake. These synthetic substrates may be useful in targeting pharmacologic agents to macrophages, and analogous compounds may target such agents to other types of cell. Macrophages play important roles in immune responses, in chronic inflammation, and in some parasitic diseases. In each case, it may be useful to deliver pharmacologic agents selectively to macrophages, particularly subpopulations of macrophages such as those in inflamed joints or in lymph nodes. We are attempting to determine whether the carbohydrate-specific glycoprotein uptake system of macrophages (1) can be used for macrophage-specific drug targeting.Mammalian macrophages specifically bind and internalize glycoproteins bearing exposed residues of mannose, N-acetylglucosamine, glucose, and L-fucose (1-3). The substrate glycoproteins are degraded within lysosomes, while the receptor component of the uptake system apparently functions repeatedly without being degraded (4). A protein that seems to be the receptor for this system has been isolated from rabbit liver (5) and serum (6) and from rat liver (7) and lymph nodes (8). The components and operation of the macrophage glycoprotein uptake system are quite similar to those of the galactose-specific mammalian hepatocyte system described by Ashwell, Morell, and coworkers (9-11). Analogous systems with other sugar specificities have also been found (12)(13)(14)(15) In the hope that these glycoprotein uptake systems may be useful in tissue-specific delivery of pharmacologic agents, we have...

Lysine-based Cluster Mannosides That Inhibit Ligand Binding to the Human Mannose Receptor at Nanomolar Concentration

“…Since preliminary binding studies revealed that the affinity of 4-aminophenyl-␣-D-mannopyranoside is only slightly higher than that of ␣-D-mannose (K i (app) ϭ 1.8 mM, log(IC 50 ) ϭ Ϫ2.48 Ϯ 0.01; data not shown), we assume this to be unlikely. An alternative explanation is that the (p-hydroxyanilino)carbamide spacer is longer (2 Å) than the thioproprionyl spacer used by Robbins et al (29). Recent studies by Biessen et al (31) have already emphasized the relevance of optimal spacing of terminal glycosides to achieve avid recognition by a comparable eukaryotic lectin, the asialoglycoprotein receptor.…”

“…Oshimi et al (28) demonstrated that the affinity of tris(hydroxymethyl)-based mannosides was only slightly higher than that of bi-and monosubstituted analogues, which was attributed to the suboptimal valency and spacing of the terminal mannosyl groups (ϳ9 Å) within the mannose clusters. The di-, tri-, and tetramannosides synthesized by Robbins et al (29) displayed higher, yet micromolar, affinities for the mannose receptor. Since the latter mannosides contained the same oligolysine backbone as our mannosides, their lower affinity is probably caused by differences in the chemical nature and the length of the spacers connecting the terminal 4.…”

“…Competition studies of 125 I-t-PA binding to endothelial liver cells confirmed the nanomolar affinity of M 6 L 5 . Previous efforts to synthesize low molecular weight ligands for the mannose receptor yielded compounds with 1000-fold lower, micromolar affinities for the mannose receptor (15,28,29). Oshimi et al (28) demonstrated that the affinity of tris(hydroxymethyl)-based mannosides was only slightly higher than that of bi-and monosubstituted analogues, which was attributed to the suboptimal valency and spacing of the terminal mannosyl groups (ϳ9 Å) within the mannose clusters.…”

Mannosylated self-assembled structures for molecular confinement and gene delivery applications

“…Several glycosylated lysine and lysine containing peptides are reported in the literature [19][20][21] and more specifically, the interaction of 3 and its galactose analog with macrophages and HepG2 cells has been described [22,23]. This background provided us an impetus to investigate self-assembly properties of mannosylated 3.…”

Imiglucerase in the treatment of Gaucher disease: a history and perspective

“…However, the seminal discovery of the mannose-binding lectin of the macrophage by Philip Stahl et al109 at Washington University, St Louis, MI, was the critical breakthrough. This revelation focused attention on delivery of proteins to be explored for therapeutic purposes in Gaucher disease – in which the prime cellular target is the eponymous macrophage 110. In vivo and laboratory studies also carried out at this time in St Louis by William Sly and colleagues (see Achord et al111) with different glycoforms of acid β-glucuronidase, which is deficient in the lysosomal disease mucopolysaccharidosis type 7 (Sly disease), led to the practical realization that the mannose/lectin receptor could be utilized efficiently to target human glucocerebrosidase to macrophages in Gaucher disease after modification of its carbohydrate residues.…”