title: The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.
abstract: The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.
authors.affiliation: Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
authors.forename: R A
authors.initials: RA
authors.lastname: DeLa Cadena
authors.forename: R W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions; D000911:Antibodies, Monoclonal; D001777:Blood Coagulation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005172:Factor XI / Q000378:metabolism; D006801:Humans; D007616:Kaolin / Q000378:metabolism; D007704:Kininogens / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D007705:Kinins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011498:Protein Precursors / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013499:Surface Properties; D015032:Zinc / Q000494:pharmacology*
chemical_list: D000838:Anions; D000911:Antibodies, Monoclonal; D007704:Kininogens; D007705:Kinins; D010446:Peptide Fragments; D011498:Protein Precursors; C046022:prekininogens; D007616:Kaolin; D005172:Factor XI; D015032:Zinc