Alpha-1 acid glycoprotein reduces hepatic leukocyte recruitment in murine models of either early endotoxemia or early sepsis.

OBJECTIVE: To characterize the effect of systemically administered AGP on early leukocyte recruitment in the livers of endotoxemic or septic mice and to determine whether this is influenced by LPS sequestration. METHODS: Endotoxemia was induced in C57Bl/6 mice via intraperitoneal injection of LPS. Sepsis was induced in mice by cecal ligation and perforation. AGP (165 mg/kg) or saline (20 mL/kg) or HAS (200 mg/kg) was administered immediately after surgery or LPS injection and the hepatic microcirculation was examined by intravital microscopy at four hour. RESULTS: Leukocyte adhesion in the PSV was reduced by treatment with AGP in mice subjected to either LPS or CLP protocols compared to either saline or HAS treatment. AGP-treated mice also had significantly higher sinusoidal flow in both models. Pre-incubation of LPS with AGP reduced the ability of LPS to recruit leukocytes to the liver microcirculation. CONCLUSIONS: AGP was more effective in limiting hepatic inflammation and maintaining perfusion than saline or HAS, in both endotoxemic and septic mice. AGP sequestration of LPS may contribute to its anti-inflammatory effects.

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.

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.

Comparison of methods for the purification of alpha-1 acid glycoprotein from human plasma.

Alpha-1 acid glycoprotein (AGP) is a highly glycosylated, negatively charged plasma protein suggested to have anti-inflammatory and/or immunomodulatory activities. Purification of AGP could be simplified if methods that exploit its high solubility under chemically harsh conditions could be demonstrated to leave the protein in its native conformation. Procedures involving exposure of AGP to hot phenol or sulphosalicylic acid (SSA) were compared to solely chromatographic methods. Hot phenol-purified AGP was more rapidly cleared from mice in vivo following intravenous injection than chromatographically purified AGP. In contrast, SSA-purified AGP demonstrated an identical in vivo clearance profile and circular dichroism spectrum to chromatographically purified AGP. Similarly, no differences in susceptibility to enzymatic deglycosylation or reactivity with Sambucus nigra lectin were detected between AGP purified via the two methods. Incorporation of the SSA step in the purification scheme for AGP eliminated the need for a large (4 mL resin/mL of plasma) initial chromatographic step and simplified its purification without causing any detectable distortion in the conformation of the protein. Confirmation that this procedure is nondenaturing will simplify AGP purification and investigation of its possible biological roles in laboratory animals.

A covalently linked recombinant albumin dimer is more rapidly cleared in vivo than are wild-type and mutant C34A albumin.

Mammalian albumins are abundant plasma proteins that exhibit a relatively slow terminal clearance. For this reason they have been fused to potentially therapeutic proteins with rapid terminal clearance to produce fusion proteins with more desirable clearance profiles. A disulfide-linked albumin dimer has been described, but its abundance and stability in plasma are uncertain. To determine whether an obligatory albumin dimer incapable of dissociation would clear less rapidly than monomeric albumin, we expressed 3 recombinant rabbit serum albumin (RSA) polypeptides: H6RSA, RSA modified by the addition of an N-terminal hexahistidinyl tag; H6RSA(C34A), H6RSA with a single cysteine (Cys) 34-to-alanine (Ala) substitution (C34A); and DiRSA, H6RSA(C34A) joined by way of its C-terminus to RSA(C34A) through an intervening hexaglycine spacer. The C34A mutation was introduced to eliminate the possibility of disulfide bond-mediated dimerization. We expressed the proteins with the use of the yeast Pichia pastoris and purified them using nickel-chelate, ion exchange, and gel-filtration chromatography. After radioiodination and injection into rabbits, H6RSA and H6RSA(C34A) exhibited indistinguishable terminal catabolic half-lives (4.9 +/- 0.7 and 4.8 +/- 0.5 days, mean +/- SD), whereas that of DiRSA was reduced to 3.0 +/- 0.3 days (p<.05). The three proteins circulated in intact form, and their distributions in liver, lung, kidney, heart, and spleen did not differ 24 hours after injection. Although more DiRSA than H6RSA(C34A) was present in urine, in both cases it was in acid-soluble form. Ethyl palmitate treatment reduced the relative acceleration of DiRSA clearance compared with that of H6RSA(C34A), suggesting a role for the reticuloendothelial system in the differential clearance of the larger protein. Our results suggest that an albumin fusion protein should include only a single copy of albumin; that if the fusion protein exceeds a certain size, it may not acquire the slow clearance profile of native albumin; and that albumin dimerization through Cys34 probably does not contribute substantially to albumin metabolism in vivo.