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Histidine-rich glycoprotein (HRG) is one of the major plasma proteins and thought to function in blood coagulation, fibrinolysis, and innate immune systems. The amino acid sequence of HRG revealed a multidomain structure consisting of cystatin-like domains 1 and 2, a Pro-rich domain 1, a His-rich domain, a Pro-rich domain 2, and a C-terminal domain. Broad ligand-binding properties of HRG are involved in the multivalent functions of HRG. Among various functions of HRG, its interactions with heparin/heparan sulfate, fibrinogen, and plasminogen are thought to be intimately related to its roles in the coagulation and fibrinolytic systems. Recent studies on these topics are mainly reviewed in this article. The newly disclosed abilities of HRG in angiogenesis, its antibacterial effect, its activation of T-cell lines in cooperation with Concanavalin A, and the identification of a putative receptor for HRG on T cell lines are also described., (© Thieme Medical Publishers.)

Naofen has recently been identified from the rat brain/spinal cord cDNA library as a substance reactive against an anti-shigatoxin (Stx)-2 antibody. Naofen mRNA is composed of 4620 nucleotides and encodes 1170 amino acids. Naofen contains four WD-repeat domains in its N-terminus and is ubiquitously distributed in many tissues of the rat. Tumor necrosis factor (TNF)-alpha enhanced the expression of naofen mRNA in HEK293 cells in a dose-dependent manner. Furthermore, naofen siRNA, which predominantly knocked down the expression of naofen mRNA, significantly reduced both TNF-alpha-induced caspase-3 activation and apoptosis in HEK293 cells. Overexpression of naofen in HEK293 cells (FLAG-NF) spontaneously induced caspase -3 activation and apoptosis, and showed extremely high susceptibility to TNF-alpha-induced apoptosis. These results indicated that naofen may function as a novel modulator activating caspase-3, and promoting TNF-alpha-stimulated apoptosis., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcgamma receptors do not participate in this cellular event. In the present study, we identified the alpha-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of alpha- and beta-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the beta-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A.

Histidine-rich glycoprotein (HRG) promoted the adhesion and morphological changes of human T-cell line MOLT-4 in a Con A-dependent manner. This morphological change-promoting activity was specific for HRG and the Arg23-Lys66 glycopeptide from human HRG. The carbohydrate chain at Asn45 was essential for this activity. The morphological changes of MOLT-4 cells caused by HRG and Con A (HRG/Con A) were not inhibited by phosphatidylinositol 3-kinase inhibitor, wortmannin or LY294002, while the changes by Con A alone were completely inhibited by these reagents, suggesting that HRG/Con A cooperate to activate leukocytes via a signaling pathway distinct from that by Con A alone. The morphological changes by Con A were associated with pseudopodia like structure. On the other hand, the morphological changes caused by HRG/Con A were associated not only with pseudopodia like structure but also with an increase of the F-actin-rich surface protrusions. Wortmannin inhibited only the formation of pseudopodia like structure.

Naofen (GenBank accession no. EF613262), a newly found intracellular protein in the WD-repeat-2 protein family, has been cloned as an anti-verotoxin II antibody immunoreactive substance, and the nucleotide- and amino acid-sequences have been clarified. The present study was undertaken to evaluate the roles of naofen especially in carbon tetrachloride (CCl4-induced cirrhosis model of rats, also in partial hepatectomy. Naofen mRNA expressions were observed from the early phases of cirrhosis development and during regenerative phases after partial hepatectomy, more remarkable in the former. Naofen immunoreactive fragments located in the vascular endothelial cells and peri-vascular spaces in normal livers especially in Glisson's areas, being strongly stained in the connective tissues 8 weeks after starting CCl4-injections, besides in the cytoplasm of hepatocytes in pseudo-lobules. In contrast, partial hepatectomy caused a small increase of naofen expressions in the whole hepatocytes, and significantly in the endothelial cells of portal veins and hepatic arterioles. Furthermore, in parallel to the degree of naofen mRNA and protein expressions, the rates of double-nuclei cells to total hepatocytes in the Glisson's areas increased in both cirrhosis and partial hepatectomy, suggesting a relationship between naofen expression and mitosis. In in-vitro studies with cell lines, vascular endothelial growth factor, a cell proliferation stimulant, increased the naofen mRNA expressions in HepG(2) cell lines, whereas paclitaxel, a cytotoxic anti-cancer drug, diminished them in NRK52E, both concentration-dependently. These results indicated that naofen immunoreactive fragments play an important role in the cell proliferation, relevant for analyzing the regenerative phases during cirrhosis developments and after partial hepatectomy.

Histidine-rich glycoprotein (HRG) is a serum protein belonging to the cystatin superfamily. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain because of a lack of rigorous testing in an animal model. We generated mice lacking the translation start point of exon 1 of the Hrg gene, effectively resulting in a null mutation (Hrg-/-). The mice were viable and fertile but had no HRG in their blood. Antithrombin activity in the plasma of Hrg-/- mice was higher than in the plasma of heterozygous Hrg+/- or wild-type Hrg+/+ mice. The prothrombin time was shorter in Hrg-/- mice than in Hrg+/- and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg-/- mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg-/- mice was higher than in Hrg+/+ mice. These findings suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivo.

Accumulating evidence shows that collagen plays important roles in many biological systems by interacting with various proteins. The major limitation to determining protein-binding sites on collagen has been a lack of useful methods. We have developed a new strategy for mapping these sites using a photoreactive cross-linker, APDP. A unique -SH group can be introduced into collagen in the vicinity of the protein-binding sites by DTT reduction of the SS bond in the cross-linked product. To identify the sites of cross-linking in collagen, the strategy used was as follows: (i) derivatization of the free -SH group with fluorescein-5-maleimide (FM); (ii) fragmentation of collagen with an appropriate collagenase; (iii) separation of FM-labeled fragments by two-dimensional diagonal electrophoresis; and (iv) detection of cross-linked partners of collagen-binding protein as fluorescent spots under UV light. This strategy can be used to determine the binding sites of various proteins on collagen.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) superfamily, possesses anti-angiogenic and neurotrophic activities. PEDF has been reported to bind to extracellular matrix (ECM) components such as collagens and glycosaminoglycans (GAGs). In this study, to determine the binding sites for collagens and GAGs, we analyzed the interaction of recombinant mouse PEDF (rPEDF) with collagen I and heparin. By utilizing residue-specific chemical modification and site-directed mutagenesis techniques, we revealed that the acidic amino acid residues on PEDF (Asp(255), Asp(257), and Asp(299)) are critical to collagen binding, and three clustered basic amino acid residues (Arg(145), Lys(146), and Arg(148)) are necessary for heparin binding. Mapping of these residues on the crystal structure of human PEDF (Simonovic, M., Gettins, P. G. W., and Volz, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11131-11135) demonstrated that the collagen-binding site is oriented toward the opposite side of the highly basic surface where the heparin-binding site is localized. These results indicate that PEDF possesses dual binding sites for different ECM components, and this unique localization of ECM-binding sites implies that the binding to ECM components could regulate PEDF activities.

Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimer's disease genes and by Abeta amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimer's disease targeting neuroprotection.

The proband, a 76-year-old woman, suffered from dural arteriovenous fistula. Her plasma histidine-rich glycoprotein (HRG) level was 50% of the normal level. A low level of plasma HRG was also found in her third daughter. A single nucleotide substitution of T to C was found at nucleotide position 11,438 in exon 6 of the HRG gene from the proband, converting Cys223 to Arg in the second cystatin-like domain. The same mutation was also identified in her third daughter, but not in the other four family members having normal HRG levels or in 50 unrelated healthy Japanese individuals. Expression studies in BHK cells showed that substantial intracellular degradation of the mutant occurred and only about 40% of the recombinant HRG mutant was secreted. These results indicate that congenital HRG deficiency caused by a substitution of Cys223 to Arg is hereditary in this family.

We reported the first case of a congenital histidine-rich glycoprotein deficiency (HRG Tokushima) in which substitution of Gly85 with Glu (G85E) in the first cystatin domain resulted in intracellular degradation and a low plasma level of HRG [Shigekiyo, T. et al. (1998) Blood 91, 128-133]. Recently, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second cystatin domain. To investigate the molecular and cellular bases of these deficiencies, we expressed these HRG mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG was completely secreted during 4-h chase periods, both the G85E and C223R mutants were only partially secreted and primarily degraded within the cells. The intracellular degradation of the C223R mutant was almost completely inhibited in the presence of a proteasome inhibitor, lactacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells decreased during the chase periods even in the presence of the proteasome inhibitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, although proteasome-specific inhibitor lactacystin and one of the cysteine protease inhibitors, E-64-d, prevented the intracellular degradation. These results suggested that intracellular degradation of G85E HRG occurred to some extent through a hitherto unknown mechanism. Similar studies involving recombinant mutants in which Gly85 or Cys223 was replaced with several other amino acids revealed that proteins with mutations leading to the destruction of the predicted b-sheet structure of the cystatin domains were eliminated by the intracellular quality control system.

The tail bud comprises the caudal extremity of the vertebrate embryo, containing a pool of pluripotent mesenchymal stem cells that gives rise to almost all the tissues of the sacro-caudal region. Treatment of pregnant mice with 100 mg/kg all-trans retinoic acid at 9.5 days post coitum induces severe truncation of the body axis, providing a model system for studying the mechanisms underlying development of caudal agenesis. In the present study, we find that retinoic acid treatment causes extensive apoptosis of tail bud cells 24 h after treatment. Once the apoptotic cells have been removed, the remaining mesenchymal cells differentiate into an extensive network of ectopic tubules, radially arranged around the notochord. These tubules express Pax-3 and Pax-6 in a regionally-restricted pattern that closely resembles expression in the definitive neural tube. Neurofilament-positive neurons subsequently grow out from the ectopic tubules. Thus, the tail bud cells remaining after retinoic acid-induced apoptosis appear to adopt a neural fate. Wnt-3a, a gene that has been shown to be essential for tail bud formation, is specifically down-regulated in the tail bud of retinoic acid-treated embryos, as early as 2 h after retinoic acid treatment and Wnt-3a transcripts become undetectable by 10 h. In contrast, Wnt-5a and RAR-gamma are still detectable in the tail bud at that time. Extensive cell death also occurs in the tail bud of embryos homozygous for the vestigial tail mutation, in which there is a marked reduction in Wnt-3a expression. These embryos go on to develop multiple neural tubes in their truncated caudal region. These results suggest that retinoic acid induces down-regulation of Wnt-3a which may play an important role in the pathogenesis of axial truncation, involving induction of widespread apoptosis, followed by an alteration of tail bud cell fate to form multiple ectopic neural tubes.

Genomic DNA libraries were screened for the human histidine-rich glycoprotein (HRG) gene and a sequence of 15,499 nucleotides was determined. The gene is composed of 7 exons and 6 introns, and all the exon-intron boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Each of cystatin-like domains I and II of HRG is encoded by three exons, exons I to III and exons IV to VI, respectively, like those of other members of the cystatin superfamily. The entire C-terminal half of the molecule is encoded by the largest exon, VII. The first 103 nucleotides of the cDNA sequence reported for human HRG [Koide, T., Foster, D., Yoshitake, S. , and Davie, E.W. (1986) Biochemistry 25, 2220-2225] could not be found in the determined gene sequence. A homology search of this sequence against a database showed the complete matching to a part of the yeast mitochondrial DNA encoding 21S ribosomal RNA. Rapid amplification of cDNA 5' ends (5'-RACE) analysis revealed that the cDNA has multiple 5'-ends and that a possible starting point is nucleotide 104 of the reported cDNA sequence. These results suggest that the first 103 nucleotides of the cDNA sequence reported for human HRG originated from yeast mitochondrial DNA and were incidentally incorporated into the HRG cDNA in the process of the construction of a cDNA library. Various fragments obtained on restriction endonuclease digestion of the 5'-noncoding region of the HRG gene were ligated to the chloramphenicol acetyltransferase (CAT) gene and then transfected into HepG2 and 293 cells to analyze the promoter activity. The sequence between -262 and -21 from the putative translation initiation site supported the expression of CAT in HepG2 cells but not in 293 cells, suggesting that this segment promotes the liver-specific transcription of the human HRG gene.

Previously, we found the first congenital deficiency of histidine-rich glycoprotein (HRG) in a Japanese woman with thrombosis. To elucidate the genetic basis of this deficiency, we first performed Southern blot analysis and found no gross deletion or insertion in the proband's HRG gene. We then examined the nucleotide sequences of all seven exons of the proband's HRG gene. A single nucleotide substitution, G to A at nucleotide position 429, which mutates Gly85 to Glu in the first cystatin-like domain, was found in exon 3 in 13 of 22 amplified clones. This mutation generates a unique Taq I site. Exon 3 was amplified from the proband, her family members, and 50 unrelated normal Japanese individuals, and Taq I fragmentation was examined. Fragmentation of exon 3 was observed in one allele of the genes from the proband and the family members who also have decreased plasma levels of HRG. Fifty unrelated normal Japanese individuals had a normal HRG gene, indicating that the G to A mutation is not a common polymorphism. To elucidate the identified mutation as a cause for the secretion defect of HRG in the proband's plasma, we constructed and transiently expressed the recombinant Tokushima-type HRG mutant (Gly85 to Glu) in baby hamster kidney (BHK) cells, and examined an intracellular event of the mutant protein. The results showed that only about 20% of the Tokushima-type HRG was secreted into the culture medium, and intracellular degradation of the mutant was observed. Thus, the present study strongly suggests that the HRG deficiency is caused by intracellular degradation of the Gly85 to Glu mutant of HRG in the proband.

Histidine-rich glycoprotein (HRG) is structurally related to the alpha2-HS glycoprotein/fetuin family of mammalian plasma proteins; both belong to the cystatin superfamily of proteins. We expressed recombinant human HRG and alpha2-HS in Sf9 insect cells for functional analysis. Recombinant HRG bound heparin and fibrinogen while alpha2-HS did not. Both proteins inhibited the formation of apatite, recombinant HRG (IC50 approximately 1 microM) with 2-fold lower molar activity than alpha2-HS (IC50 approximately 0.5 microM). The inhibition in vitro of apatite formation suggests a new function for plasma HRG protein, inhibition of phase separation in blood vessels.

Meiotic recombinations in the proximal region of the mouse major histocompatibility complex (MHC) are clustered within certain segments of chromosome, known as hotspots. In this study, we found that one of such hotspots, previously mapped between the Pb and Ob genes, is located very close to the 3' end of the Lmp2 gene, which encodes a subunit of a proteolytic proteasome. To analyze the molecular basis of the site specificity of hotspots, we examined the structure of the chromatin around this Lmp2 hotspot and another one located in the MHC class II Eb gene, by monitoring DNase I-hypersensitive sites (DHSSs) of the chromatin. DHSSs were detected at the both hotspots in the somatic cells. In the meiotic cells, DHSS was detected within the Eb hotspot, as previously reported, but not in the Lmp2 hotspot. Thus, open structure of chromatin during meiosis, as monitored by hypersensitivity to DNase I, is not a general feature of mouse recombinational hotspots, contrasting the case of the lower eukaryote, S. cerevisiae, in which hotspots are always associated with DHSSs.

Two forms of histidine-rich glycoprotein (HRG) were detected on SDS-PAGE by silver staining and immunoblotting after isolation of the protein from pooled plasma using immuno-affinity chromatography followed by chromatography with heparin-Sepharose. Both forms were single-chain molecules and the apparent molecular weights of form 1 and form 2 were 77 kD and 75 kD respectively. Mendelian inheritance of both HRG forms was observed in four families with 24 informative meioses, strongly suggesting that the two forms are encoded by different alleles. The frequency of form 1 and form 2 in a group of 36 individuals was 0.35 and 0.65 respectively. The difference between the two molecular variants was studied by direct sequence analysis of amplified exons of the HRG gene from 6 individuals who were homozygous either for form 1 or form 2. Five amino acid polymorphisms in three different exons were observed: Ile/Thr in exon4; Pro/Ser in exon 5; His/Arg, Arg/Cys and Asn/Ile in exon 7. Analysis of these polymorphisms in 20 volunteers showed that only the Pro/Ser polymorphism at position 186 in exon 5 was coupled to the form of the HRG protein. Ser was found in form 1 and Pro in form 2. The presence of Ser at position 186 introduces a consensus sequence for a N-glycosylation site (Asn-X-Ser/Thr). By removing N-linked sugars with N-glycanase, it could be demonstrated that the difference between the two forms of HRG is caused by an extra carbohydrate group at Asn 184 in form 1.

Previously we reported that ornithine decarboxylase (ODC) is degraded ATP-dependently by the 26 S proteasome in the presence of antizyme (AZ), an ODC inhibitor (Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992) Nature 360, 597-599). Here we examined the cleavage of ODC by the 26 S proteasome. When ODC purified from ODC-overproducing cells was incubated with the 26 S proteasome and with AZ fused with maltose-binding protein (MBP) in the presence of ATP, ODC was degraded specifically without appreciable breakdown of MBP-AZ. The major degradation products of ODC, which were separated by high performance liquid chromatography on a reverse-phase column, were identified by N-terminal amino acid sequencing. The 26 S proteasome generated a variety of short peptides of 5-11 amino acid residues derived from regions throughout the ODC sequence. No detectable amounts of free amino acid residues were produced, indicating endoproteolytic degradation of ODC by the 26 S proteasome. Their major sites for cleavage of ODC by the 26 S proteasome were on the carboxyl sides of neutral/hydrophobic amino acid residues, but a few were on those of acidic or basic amino acid residues. These results demonstrate that the 26 S proteasome causes exhaustive endoproteolysis of the naturally occurring short-lived protein ODC in a multicatalytic and ATP-dependent manner.

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). the heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca(2+)-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparin-neutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+. In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.

Histidine-rich glycoprotein (HRG) is a monomeric plasma glycoprotein involved in the modulation of coagulation and fibrinolysis. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HRG-specific cDNA probe, the HRG gene was assigned to chromosome 3. One hybrid that was known to contain only a segment of chromosome 3 also reacted positively with the HRG probe. Hybridization analysis with a set of chromosome 3-specific probes showed that the segment of chromosome 3 present in this hybrid is missing the region pter-p14, which indicates that HRG is not located in this region. No restriction fragment length polymorphisms were detected for HRG with 10 commonly used restriction enzymes.

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.

The human chromosomal histidine-rich glycoprotein (HRG) gene has been isolated and its molecular structure was partially characterized. The gene is approximately 11 kb in length and contains nine exons and eight introns. Locations of the introns in HRG gene coding for cystatin domains are essentially identical with those of cystatin SN, SA and C, and kininogen genes. These results provide direct evidence that HRG belongs to a supergene family that includes cystatin SN, SA and C, and kininogen, and also demonstrate high conservation of the intron-exon organization among this supergene family.

The plasma proteins alpha 2-plasmin inhibitor and histidine-rich glycoprotein were compared directly with respect to their effectiveness in inhibiting the binding of plasminogen to fibrin under the same experimental conditions. At their physiological concentrations, the presence of alpha 2-plasmin inhibitor more markedly decreased the binding of plasminogen to fibrin than histidine-rich glycoprotein. Significance of these findings in inhibition of fibrinolysis is discussed.

A high degree of sequence homology has been found between the N-terminal region of histidine-rich glycoprotein (HRG) and that of antithrombin III (AT III) where the putative heparin-binding site of AT III is located. The amino acid residue at the position corresponding to Arg-47 of AT III that is essential for the heparin-binding was also arginine (Arg 23 and 78) in the homologous sequences of HRG. These observations strongly suggest that the heparin-binding sites of HRG and AT III are evolutionarily related. There was no apparent sequence similarity between the remaining about 70% portions of the two proteins.

The simple and simultaneous purification of histidine-rich glycoprotein (HRG) and antithrombin III (AT III) from human plasma and gross structural characterization of HRG have been performed. The purification method consists of two chromatographic procedures using heparin-agarose and DEAE-Sephadex. The yields of HRG and AT III were 22 mg and 70 mg, respectively, from 1 liter of plasma. The purified HRG is a single-chain polypeptide with a molecular weight (Mr) of 75,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, indicating it was the native form of this protein. Cyanogen bromide cleavage of HRG, followed by analysis of the amino acid composition and determination of the amino-terminal sequence of each purified cyanogen bromide fragment established that the gross structure of HRG consisted of three cyanogen bromide fragments; an amino-terminal CN-50 kDa fragment (Mr 50,000) and a carboxy-terminal small fragment of eight amino acids, and a CN-30 kDa fragment (Mr 30,000) between them. As to the amino acid composition of the CN-30 kDa fragment, it had an unusually high content of histidine (25 mol%), suggesting the presence of a histidine-rich region(s) in the carboxy-terminal half of the molecule. These results together with our previous results (Koide, T., Odani, S., & Ono, T. (1982) FEBS Lett. 141, 222-224) and those of Morgan (Morgan, W.T. (1985) Biochemistry 24, 1496-1501) imply that HRG is composed of at least two domains with distinct functional properties; i.e. an amino-terminal domain with heparin-binding ability and a carboxy-terminal domain with heme- and divalent metal-binding abilities.

A new member of the cystatin superfamily is introduced. Human plasma histidine-rich glycoprotein (HRG) was found to contain 2 cystatin-like sequences in tandem in the N-terminal region. Domain 1 (residues 1-112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113-225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.