Occurrence of beta-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains.

Abstract

Vitamin K-dependent bovine protein S has been shown to contain a posttranslationally hydroxylated asparagine within a conserved sequence in three of its epidermal growth factor (EGF)-like domains. In a review of amino acid sequences deduced from cDNA data, we have observed that a conserved sequence containing a potential asparagine hydroxylation site exists within EGF-like domains of a variety of functionally diverse proteins. We have studied a number of these and report the presence of erythro-j8-hydroxyasparagine (e-fiHyn) in three non-vitamin K-dependent proteins: the plasma complement proteins COr and Cls (where overbar indicates activated form) and the urinary protein uromodulin. For each protein, e-fiHyn was identified in enzyme digests following the initial observation of erythro-18-hydroxyaspartic acid (e-I3Hya) in acid hydrolysates of the proteins. efiHya and e-fiHyn residues are detected by a postcolumn derivatization cation-exchange HPLC method herein described. HPLC isolation of th presumptive e-fiHyn residue from enzyme digests of intact COr allowed confirmation of its structure by GC/MS.Based upon available cDNA sequence data and observation of e-f3Hya in acid hydrolysates, we suggest other proteins in which e-fiHyn may occur.Recent cDNA sequence data demonstrate the existence of common structural domains in widely diverse proteins (1-3). One such example, the epidermal growth factor (EGF)-like repeat, contains about 40 amino acids, including six cysteine residues that give rise, through a particular set of disulfide bridges, to a specific triple-looped structure. The EGF-like domain is found in a number of plasma proteins as well as extracellular portions of plasma membrane protein receptors (1-3). In the most N-terminal EGF-like domain of all vitamin K-dependent coagulation proteins (with the exception of prothrombin, which lacks EGF-like domains) a single conserved aspartic acid residue is posttranslationally hydroxylated to erythro-/3-hydroxyaspartic acid (e-PHya) (4 DL-e-pHya and DL-threo-f3Hya (t-,BHya) (18) and DL-e-,BHyn and DL-t-j3Hyn (19) were synthesized as described.HPLC. The system used was an adaptation of the postcolumn amino acid analysis system described by Fernlund and Stenflo (11). An analytical column (10 cm x 0.46 cm i.d.)Abbreviations: i3Hya, ,-hydroxyaspartic acid; ,3Hyn, ,8-hydroxyasparagine; EGF, epidermal growth factor; C, complement; e-, erythro-; t-, threo-; Gla, 'y-carboxyglutamic acid; TM, thrombomodulin; TSP, thrombospondin; LDL, low density lipoprotein; Me3Si, trimethylsilyl. §To whom reprint requests should be addressed. 7856The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Interaction of Factor IXa with Factor VIIIa

“…1). Plasma factor IX and each recombinant protein had similar Gla content (10.7-11.4 residues) as measured by the technique of Przysiecki et al (43). The amino-terminal sequence of each mutant protein was also 5 One unit/ml of factor VIII after IIa activation contains ϳ0.7 nM factor IXa binding sites (see "Results and Discussion").…”

“…Circulating factor IX consists of an amino-terminal ␥-carboxyglutamic acid (Gla) 1 domain (residues 1-40), a short hydrophobic segment (residues [41][42][43][44][45][46], two epidermal growth factor (EGF)-like domains (EGF1 residues 47-84 and EGF2 residues 85-127), an activation peptide region (residues 146 -180), and the carboxyl-terminal serine protease domain (residues 181-415). Based upon the crystal structure of the Gla domain of factor VIIa (8), the Ca 2ϩ binding properties of factor X, 2 and the NMR structure of the Gla domain of factor IX (10), it would appear that this domain in factor IX possesses several low to intermediate affinity Ca 2ϩ binding sites.…”

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

“…We predict that these type C sequences are hydroxylated on the appropriate aspartate or asparagine residue (see Figure 3) and bind Ca2>. Recently amino acid analysis has shown that uromodulin, thrombospondin and thrombomodulin (Przysiecki et al, 1987) contain detectable levels of (-hydroxyasparagine, although the exact location remains to be determined.…”

Structural Integrity of the γ-Carboxyglutamic Acid Domain of Human Blood Coagulation Factor IXa Is Required for Its Binding to Cofactor VIIIa

“…Immunoblotting was performed using a chemiluminescence detection system employing a peroxidase-conjugated polyclonal F.IX antibody. Gla analysis was performed according to the modified alkaline hydrolysis method of Price (16). Amino-terminal protein sequence analysis was performed by automated Edman degradation to confirm the mutation at position 12.…”