Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the .beta.-antithrombin III isoform with enhanced heparin affinity

Picard, Véronique; Ersdal-Badju, Eva; Bock, Susan

doi:10.1021/bi00026a026

Cited by 87 publications

(99 citation statements)

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“…It is interesting that the fully glycosylated sequons all end with threonine, whereas those that are only partially glycosylated end with serine. This agrees with the recent report that when serine is in the third position, glycosylation is less complete than in the case of threonine (19). For Asn 155 the effect of the disulfide bond discussed above may be an additional factor in reducing the level of glycosylation.…”

Section: Identification Of N-linked Glycosylation Sites In Purified Hsupporting

confidence: 92%

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Sturrock

et al. 1997

Journal of Biological Chemistry

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The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential Nlinked glycosylation sites, Asn 90 and Asn 109 , were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn 155 , Asn 337 , and Asn 586 , are partially glycosylated, whereas Asn 72 appears to be fully glycosylated. The only potential site that was not modified is Asn 620 . Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser 625 . When expressed in the presence of the ␣-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.Both forms of angiotensin-converting enzyme (ACE 1 ; EC 3.4.15.1 peptidyl-dipeptidase A) are class I transmembrane ectoenzymes (1) that have N-and O-linked oligosaccharides attached to their polypeptide chains (2, 3). Expression of ACE in human HeLa cells in the presence of tunicamycin resulted in complete inhibition of glycosylation, rapidly degraded intracellular ACE, and no enzyme released in the medium (4). An enzymatically active ACE was produced with partial glycosylation in a mutant Chinese hamster ovary (CHO) cell line (ldlD), although it was released to a lesser extent (4). Similarly, it was reported (5) that inhibitors of glucosidases I and II in the endoplasmic reticulum (ER) and mannosidase I in the cis-Golgi reduced the amount of oligosaccharide attached to human intestinal ACE and delayed protein release significantly. These data strongly suggest that glycosylation plays an important role in the membrane targeting and release of ACE, possibly by affecting the folding of the polypeptide and its recognition by a variety of enzymes in the folding and transport machineries. Recently, Sadhukhan and Sen (6) reported that mutations at individual N-linked glycosylation sites (sequons) in rabbit testis ACE (tACE) resulted in varied efficiencies in enzyme release, which suggests that N-linked glycans at each site may make diff...

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Section: Identification Of N-linked Glycosylation Sites In Purified Hsupporting

confidence: 92%

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Sturrock

et al. 1997

Journal of Biological Chemistry

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“…The N135A substitution eliminates binding heterogeneity associated with partial glycosylation of asparagine 135 (31) and directs production of a homogeneous ␤-ATIII-like parent molecule with high base-line affinity for heparin (37). Basic-to-Ala substitutions were introduced using a one-tube polymerase chain reaction mutagenesis method (38).…”

Section: Atiii Expression System and Site-directed Mutagenesis-mentioning

confidence: 99%

“…After 10 min at 37°C, reactions were subjected to non-reducing SDS-polyacrylamide gel electrophoresis and Western blotting with sheep anti-human ATIII Ig (Binding Site, Birmingham, UK) as described previously (31).…”

Section: Atiii Expression System and Site-directed Mutagenesis-mentioning

confidence: 99%

“…The naturally occurring ␤-isoform of ATIII lacks carbohydrate on asparagine 135 (30) because its NXS type consensus sequence is inefficiently glycosylated (31). The ␤-isoform binds heparin more tightly than does ␣-isoform due to effects of the Asn 135 oligosaccharide on the heparin-induced conformational change which accompanies formation of the high affinity heparin-ATIII complex (59).…”

mentioning

confidence: 99%

“…Antithrombins expressed in BHK and CHO cell lines are heterogeneously glycosylated due to nonuniformity in host cell post-translational modification processes. This kind of glycosylation heterogeneity, in addition to ␣-ATIII/␤-ATIII glycosylation heterogeneity caused by the wild type Asn 135 NXS consensus sequence (31), causes the expressed antithrombin molecules to display a broad range of affinities for heparin (32,35,36). Consequently, it is problematic to determine the heparin affinities of ATIII variants produced in these expression systems, and they are not optimal for in vitro mutagenesis studies of the ATIII heparin binding site (35).…”

mentioning

confidence: 99%

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Identification of the Antithrombin III Heparin Binding Site

Ersdal-Badju

Zuo

et al. 1997

Journal of Biological Chemistry

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The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

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Antithrombin

Bj��rk¹

2002

Wiley Encyclopedia of Molecular Medicine

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Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the .beta.-antithrombin III isoform with enhanced heparin affinity

Cited by 87 publications

References 46 publications

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Identification of the Antithrombin III Heparin Binding Site

Antithrombin

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