In vivo clearance of alpha-1 acid glycoprotein is influenced by the extent of its N-linked glycosylation and by its interaction with the vessel wall

Abstract

Alpha-1 acid glycoprotein (AGP) is a highly glycosylated plasma protein that exerts vasoprotective effects. We hypothesized that AGP's N-linked glycans govern its rate of clearance from the circulation, and followed the disappearance of different forms of radiolabeled human AGP from the plasma of rabbits and mice. Enzymatic deglycosylation of human plasma-derived AGP (pdAGP) by Peptide: N-Glycosidase F yielded a mixture of differentially deglycosylated forms (PNGase-AGP), while the introduction of five Asn to Gln mutations in recombinant Pichia pastoris-derived AGP (rAGP-N(5)Q) eliminated N-linked glycosylation. PNGase-AGP was cleared from the rabbit circulation 9-fold, and rAGP-N(5)Q, 46-fold more rapidly than pdAGP, primarily via a renal route. Pichia pastoris-derived wild-type rAGP differed from pdAGP in expressing mannose-terminated glycans, and, like neuraminidase-treated pdAGP, was more rapidly removed from the rabbit circulation than rAGP-N(5)Q. Systemic hyaluronidase treatment of mice transiently decreased pdAGP clearance. AGP administration to mice reduced vascular binding of hyaluronic acid binding protein in the liver microcirculation and increased its plasma levels. Our results support a critical role of N-linked glycosylation of AGP in regulating its in vivo clearance and an influence of a hyaluronidase-sensitive component of the vessel wall on its transendothelial passage.

Figure 1

Neuraminidase treatment of human AGP increases its clearance from the rabbit circulation. Plasma-derived AGP with (neu-AGP) or without (AGP) neuraminidase treatment was examined on a 12% SDS reducing gel, either by Coomassie Blue staining ((a), left, stained gel) or by ligand blotting with SNA ((a), right, SNA blot). The position of molecular mass markers is shown in kDa, at left. Tfn: transferrin; GST: glutathione sulfotransferase. (b) Shows the recovery (% injected dose) of acid-precipitable radioactivity in plasma at different times following injection of radioiodinated AGP (solid bar) or neu-AGP (open bar) into rabbits. *Indicates P < 0.05 versus corresponding AGP value by unpaired t-test, Welch corrected. The mean of 7 determinations ± SD is shown.

Figure 2

Forms of AGP with reduced or absent N-linked glycosylation are cleared from the rabbit circulation more rapidly than native AGP following intravenous injection. (a) Shows a Coomassie blue-stained 12% SDS reducing gel loaded with 5–10 μg per lane of human serum albumin, HSA; AGP; AGP treated with PNGase F (PNGase) in the presence of either urea (PNGase-AGP (U) or SDS (PNGase-AGP (S); or recombinant yeast-derived nonglycosylated AGP (rAGP N(5)Q). (b) Depicts the residual acid-precipitable radioactivity remaining in the plasma of rabbits after intravenous injection of the indicated radiolabeled proteins as a function of time; for each protein, plasma radioactivity is presented as the percentage of the injected radioactive dose recovered in plasma at the indicated time after injection. Each point is the mean ± SD of 6 determinations; in most instances error bars are smaller than the symbol that denotes the mean.

Figure 3

Forms of AGP with reduced glycosylation are cleared from the rabbit circulation and distribute to the kidneys more rapidly than native AGP. Plasma from rabbit injected with radioiodinated proteins (identified below (a)–(c)) was electrophoresed on reduced SDS-PAGE gels; autoradiograms of samples taken 5 and 15 minutes after injection are shown. Molecular mass markers in kDa are shown at left, and the deduced number of N-linked glycan chains at right. (d) Shows the percentage of the total radioactive dose of injected radioactivity found in the organs (identified below the panels) 30 minutes after injection of AGP (solid bars) or PNGase F-treated AGP (PNGase-AGP, open bars) or rAGP-N(5)Q (stippled bars). The mean of 6 determinations ± SD is shown. *Indicates P < 0.001 versus AGP value for the other two proteins within each organ sampled; all other comparisons to AGP values within organs are not significant.

Figure 4

Recombinant AGP glycosylated in Pichia pastoris yeast is rapidly removed from the rabbit circulation via mannose receptors. (a) Shows the radioiodinated rAGP-WT or rAGP N(5)Q remaining in the plasma of rabbits 30 minutes after injection, without (no sugar) or with coadministered α-methylmannoside (+α MM) or D-galactose (+ Galactose). The mean and SD of 5–7 determinations are shown; *indicates P < 0.05 versus the corresponding “no sugar” condition by Kruskal-Wallis test. The proportion of the two proteins remaining in plasma in the absence of added sugar was also compared; ***indicates P < 0.001 by unpaired t-test, Welch corrected. (b) (Gel) shows a Coomassie blue-stained 12% SDS reducing gel loaded with 5–10 μg per lane of AGP (plasma-derived); rAGP-WT; carboxypeptidase Y (CPY); and glutathione sulfotransferase (GST). Molecular mass marker positions, shown at left, correspond to: 100; 90; 80; 70; 60; 50; 40; 30; 25; and 20 kDa. (c) (GNA blot) is a blotted replicate of the gel shown in (b), probed with GNA lectin.

Figure 5

The disappearance of injected AGP from the plasma of mice following intravenous injection is altered by systemic hyaluronidase treatment. Radioiodinated AGP was injected into mice with (AGP + hy'ase) or without (AGP) concurrent hyaluronidase treatment, and acid-precipitable radioactivity, shown as the percentage of the dose remaining in plasma, was followed over time, n = 6 ± SD.

Figure 6

Either systemic hyaluronidase treatment or AGP administration reduces HABP binding to the vessel wall of mouse liver sinusoids in vivo. Mice were treated with hyaluronidase (Hy'ase; (b) and (e)) or without hyaluronidase (no treatment; (a) and (d)) after injection of either fluorescent HABP complex or fluorescent GS-1B₄ and visualization of liver sinusoids by confocal intravital microscopy. (c) Is an image of an experiment in which AGP intravenous injection was substituted for hyaluronidase. (f) (Mean of n = 3 ± SD) quantifies the intrasinusoidal HABP fluorescence in the experiment shown in (c) and in three additional replicates; each replicate is the mean of four intravascular samples within microscopic fields similar to that seen in (c); *signifies P < 0.05 by unpaired t-test, Welch corrected.