Comparative properties of human α‐1‐proteinase inhibitor glycosylation variants

Guzdek, A; Potempa, Jan; Dubin, Adam; Travis, James

doi:10.1016/0014-5793(90)80464-t

Cited by 25 publications

(12 citation statements)

References 17 publications

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“…Another protein in which decreased glycosylation leads to increased biological activity is erythropoetin, for which removal of one of three oligosaccharide side chains by sitespecific mutagenesis was shown to almost double the activity (Yamaguchi et al, 1991). In contrast, glycosylation variants of a plasma proteinase inhibitor related to antithrombin, aLproteinase inhibitor, had indistingishable inhibitory activities (Guzdek et al, 1990), consistent with the observations in the present work that the differences in glycosylation ofantithrombin did not affect the intrinsic rate constant of the uncatalysed or heparin-catalysed inhibition of thrombin. The observation that an increased glycosylation of recombinant antithrombin leads to a decreased affinity for heparin is in agreement with previous work indicating that the carbohydrate side chains of antithrombin affect its heparin-binding properties.…”

Section: Discussionsupporting

confidence: 92%

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Björk

Ylinenjärvi

Olson

et al. 1992

Biochemical Journal

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Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proporti6n of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

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Section: Discussionsupporting

confidence: 92%

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Björk

Ylinenjärvi

Olson

et al. 1992

Biochemical Journal

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“…The higher stoichiometric indices observed for hNS-H 6 as compared with cNS-C could reflect a lower stability of the complexes formed by human neuroserpin. Alternatively, this observation could be explained by the fact that the hNS-H 6 used for these experiments was of procaryotic origin and, thus, less stable due to a lack of glycosylation (65).…”

Section: Fig 4 Complex Formation and Inhibitory Activity Of Neurosementioning

confidence: 94%

The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin

Osterwalder

Cinelli

Baici³

et al. 1998

Journal of Biological Chemistry

130

112

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Neuroserpin is an axonally secreted serine proteinase inhibitor that is expressed in neurons during embryogenesis and in the adult nervous system. To identify target proteinases, we used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig--variable region, an Ig-enhancer, and the exon encoding the Igconstant region (C) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with C. We investigated the capability of recombinant neuroserpin to form SDS-stable complexes with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in the nervous system mediated by plasminogen activators or plasmin.Extracellular proteolysis exerted by serine proteinases has been implicated in a variety of processes in the nervous system during development and in adulthood. Among the serine proteinases recently reported to play a role in neural development and function, there are several well known proteins that had previously been found and characterized in nonneuronal functions, in particular blood coagulation and fibrinolysis. For example, tissue-type plasminogen activator (tPA) 1 and urokinase plasminogen activator (uPA) were found to be expressed in the nervous system (1, 2), and they have been demonstrated to be engaged in developmental processes such as cerebellar granule cell migration (3, 4), Schwann cell migration, and wrapping of axons (5), or neuromuscular synapse elimination (6). In the period of neurite outgrowth, plasminogen activators (PAs) have been found to be secreted at the growth cones of cultured neurons or neuronal cell lines (7,8), and they were demonstrated to modify the molecular composition of the neurites' substrata in vitro (9). In the adult nervous system, tPA is induced in the hippocampus after seizure, kindling, and long term potentiation (LTP) (10) and in the cerebellum after motor learning tasks (11), and mice lacking the gene for tPA (12) show a different form of hippocampal LTP (13,14). Furthermore, tPA has been demonstrated to be involved in excitotoxin-induced neuronal cell death in the murine hippocampus by converting locally secreted plasminogen to active plasmin (15, 16). Thrombin, which has been extensively characterized due to its important function in the blood clotting system, has been reported to be expressed in the nervous system (17). I...

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“…This in turn markedly reduces the a 1 -antitrypsin that is available to protect the lungs against proteolytic attack by the enzyme neutrophil elastase [41]. The situation is exacerbated as the Z mutation reduces the association rate between a 1 -antitrypsin and neutrophil elastase approximately fivefold [42][43][44][45]. Thus, the a 1 -antitrypsin available within the lung is not as effective as the normal M protein.…”

Section: Polymerization Of Z a 1 -Antitrypsin And Emphysemamentioning

confidence: 93%