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The crystal structure of bikunin from the inter-α-inhibitor complex: A serine protease inhibitor with two kunitz domains1

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Abstract

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 Å data. The two BPTI-like domains pack closely together and are related by an approximate 60° rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.

Introduction

Bikunin is a serine protease inhibitor that is effective against a broad range of enzymes that includes trypsin, chymotrypsin, plasmin and leukocyte elastase, as well as cathepsins B and H. It occurs at high levels in blood serum as well as in urine and has been found in other tissues. In urine it exists in its free form, while in blood it exists in both its free form and as part of the inter-α-inhibitor (IαI) complex. Bikunin has attracted a great deal of attention in the last few years due to the observation that it occurs at elevated levels in several disease states including inflammation and cancer. It has been shown, under some conditions, to effectively inhibit tumour invasion and metastasis (Kobayashi et al., 1994a) and is presently prescribed in Japan for the treatment of acute pancreatitis and haemorrhagic shock (Shikimi & Handa, 1986).

The biological activity of IαI and bikunin has been investigated by a number of laboratories and good reviews of this work are available Salier 1990, Salier et al 1996. Bikunin has been implicated in the regulation of cell growth. It has a biphasic effect on cell growth; at low concentrations it is capable of promoting growth while at high concentrations it inhibits cell growth (Perry et al., 1994). The observation that bikunin is capable of inhibiting cell growth has led to tests of its effect on tumours (Chawla et al., 1992). Experiments with highly purified bikunin have established that it inhibits tumour cell invasion in an in vitro model and spontaneous lung metastasis in an in vivo mouse model (Kobayashi et al., 1994a). A cell surface receptor binding site for bikunin, which is distinct from the protease recognition site, is known to reside in the first domain (Kobayashi et al., 1995). Bikunin receptors have been identified in some cancer cells, such as human choriocarcinoma SMT-cc1 cells and murine Lewis lung carcinoma 3LL cells (Kobayashi et al., 1994b). Apart from growth regulation, other functions have been attributed to bikunin or its domains. The C-terminal domain of the rat bikunin has been isolated from peritoneal mast cells and found to inhibit tryptase and blood coagulation factor Xa Kido et al 1988, Itoh et al 1994. It has been suggested that this domain, trypstatin, may be involved in the allergic and other inflammatory responses. In addition to its functions within the bloodstream, bikunin may have a role in urine. It has been identified as the uronic-acid-rich protein (UAP) that inhibits calcium oxalate crystallization in vitro and that has been implicated in urolithiasis (Atmani et al., 1996).

Bikunin has had a very long history in the literature and has been referred to as mingin (Astrup & Nissen, 1964), urinary trypsin inhibitor (UIT) and HI30 (Gebhard & Hochstrasser, 1986). The sequence of bikunin derived from cDNA sequencing studies shows that its gene encodes two proteins; α1-microglobulin (α1m) followed by bikunin (Kaumeyer et al., 1986). α1-Microglobulin and bikunin are separated by a short dibasic connecting peptide that is cleaved in the Golgi apparatus of the liver (Bratt et al., 1993). Of the bikunin sequences shown in Table 1, those originating from mammals are very similar to one another but differ significantly from those of lower organisms. Human bikunin, as isolated from serum or urine, consists of about 145 amino acid residues, with two segments that are homologous to bovine pancreatic trypsin inhibitor (BPTI) of the Kunitz family of inhibitors (Gebhard & Hochstrasser, 1986). There are about 21 residues that precede the first Kunitz domain (residues 22 to 77), while the second domain (residues 78 to 133) is followed by another ten residues. The protein is known to be heavily glycosylated at two sites, Ser10 and Asn45, with carbohydrate making up about 50% of the observed mass of the protein (Gebhard & Hochstrasser, 1986). The reported molecular mass varies considerably depending on the source and method of purification (Chawla et al., 1992). Much of this variation can be explained by proteolysis within the first 22 residues and carbohydrate degradation (Gebhard & Hochstrasser, 1986).

High-resolution crystal structures of a number of Kunitz family proteins have been determined. These include BPTI (PDB code 4PTI; Deisenhofer and Steigemann 1975, Wlodawer et al 1984, Wlodawer et al 1987), Alzheimer’s amyloid β-protein precursor inhibitor domain (APPI with PDB code 1AAP; Hynes et al., 1990), α-dendrotoxin (α-DTX with PDB code 1DTX; Skarzynski, 1992) and the Kunitz-type domain from the α3 chain of human type VI collagen (C5 with PDB code 1KNT; Arnoux et al., 1995). In addition, there is a medium-resolution structure of β-bungarotoxin (PDB code 1BUN) that consists of a Kunitz domain along with a phospholipase A2 domain (Kwong et al., 1995). The Kunitz domain in β-bungarotoxin binds a receptor and, like α-dendrotoxin and C5, it has no capacity to inhibit proteases. The sequences of these proteins and their alignment to BPTI are shown in Table 1. The structure of BPTI as a complex with trypsin has been determined so that the structural features of BPTI that are important for protease recognition have been identified (PDB code 2PTC; Ruhlmann et al 1973, Chen and Bode 1983). In BPTI, there are two loops that are particularly important for recognition: the first recognition loop consists of residues 11 to 19, while the second is positioned nearby and consists of residues 34 to 39. Structural comparisons reveal that these two loops are not highly conserved in different protease inhibitors, as might be expected from the individual specificities. Lys15 of BPTI is located in the middle of the first loop, and is the principal determinant of inhibitor specificity. Sequence comparisons (Table 1) indicate that the corresponding residues in human bikunin are Met36 on the first domain and Arg92 on the second domain. These assignments are consistent with experiments that show that the N-terminal domain inhibits elastase, while the C-terminal domain inhibits trypsin and plasmin (Gebhard & Hochstrasser, 1986). Recently, the structure of ornithodorin-thrombin has been reported (van der Locht et al., 1996). Ornithodorin is isolated from ticks and is a potent and highly selective thrombin inhibitor that contains two BPTI-like domains. Thrombin shows little affinity for most Kunitz-BPTI like inhibitors and it is not surprising that ornithodorin functions quite differently from BPTI. The thrombin does not bind ornithodorin at the usual “recognition loops”, which assume quite different conformations to those of BPTI (van der Locht et al., 1996). In fact, the loops point away from the enzyme in the structure of the complex.

We report here, the crystallization and three-dimensional structure of bikunin, describe the spatial arrangement of the two inhibitor domains and comment on functional implications of the structure.

Section snippets

Quality of the model

The structure of bikunin is reasonably well determined, as is evident from the refinement statistics shown in Table 2 and the definition in the electron-density difference map shown in Figure 1. The final model contains only the two BPTI-like domains. There was no electron density for residues 1 to 24 or 135 to 145. The C-terminal residues are probably disordered. The weight of evidence suggests that the N terminus has been proteolytically removed. This is consistent with the fact that crystals

Concluding Remarks

The two bikunin domains exhibit a significant number of contacts suggesting that the packing observed in the crystals is stable and would persist in solution. Sequence comparisons imply that the arrangement of domains found in bikunin is not present in other proteins with more than one BPTI module. Our structure suggests that proteases binding to domain II could be affected by the presence of domain I. This observation may explain why domain II of bikunin will inhibit factor Xa and tryptase,

Protein and crystallization

Bikunin was obtained as a freeze-dried solid from the Biochemical Factory of Nanjing University, and was used without further purification. The purity of the protein was examined using SDS/10% polyacrylamide gel electrophoresis. The most prominent feature on the gel was a diffuse collection of bands that corresponded to a molecular mass of about 30 kDa. These bands provided sufficient material for N-terminal sequence determination using an Applied Biosystems sequencer. It was found that the

Acknowledgements

We thank Dr Ray Withers for help in displaying structures of calcium oxalate and Mr Boda Zhang for providing the protein for this study. We acknowledge ANU supercomputer Facility for a grant of time.

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