Review
Bikunin — not just a plasma proteinase inhibitor

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Abstract

Bikunin is a plasma proteinase inhibitor that has received little attention in the past, probably because its activity towards various proteinases was found to be relatively weak in early work. It was recently discovered, however, that bikunin effectively inhibits a proteinase that seems to be involved in the metastasis of tumour cells — cell surface plasmin — and that a fragment of bikunin inhibits two proteinases of the coagulation pathway — factor Xa and kallikrein. Furthermore, it has been found that bikunin has other properties, such as the ability to modulate cell growth and to block cellular calcium uptake. Most of the bikunin in the blood occurs as a covalently linked subunit of the proteins pre- and inter-α-inhibitor. In this form bikunin lacks some of its known activities, and there is evidence that its release by partial proteolytic degradation may function as a regulatory mechanism. Although the physiological function of bikunin still remains to be established, current data suggest that this protein plays a role in inflammation. Further studies could therefore lead to results of therapeutical value.

Introduction

In 1909 it was reported that urine of patients suffering from certain pathological conditions had the capacity to inhibit trypsin [1]; it was later found that normal urine also has this activity but of a much lower level [2]. In 1955 the protein responsible for most of the antitryptic activity in urine was isolated [3], and it was subsequently often referred to as the urinary trypsin inhibitor (UTI) [3], [4]. The protein was found to remain soluble and active after treatment with moderate concentrations of strong acids [5], [6] and was therefore also called the acid stable trypsin inhibitor (ASTI) [7]. Furthermore, it was discovered that when the acid insoluble serum proteins were digested with trypsin, a polypeptide identical to the urinary trypsin inhibitor was released, which was named HI-30 [8], [9]. Even more names for the same protein were used in the literature, such as mingin [10], urinastatin and ulinastatin [11]. To eliminate the confusion caused by the existence of many different names, a number of workers in the field agreed in 1990 to use a new name — bikunin [12] — which will be used throughout this article.

Section snippets

Polypeptide

When the amino acid sequence of bikunin — isolated from human serum — was elucidated [13], it became clear that the protein contains two proteinase inhibitor domains of the Kunitz type (see Fig. 1; green and red circles, respectively); hence the newly introduced name bikunin [12]. A Kunitz inhibitor has a molecular mass of about 7 kDa and three disulphide bonds arranged in a characteristic fashion. In addition to the two Kunitz domains, bikunin contains a short connecting peptide as well as N-

Free bikunin

The cDNA for bikunin was cloned in 1986 from a human liver cDNA library. Its sequence showed that it codes for a precursor also containing alpha1-microglobulin [40], a 25 kDa plasma protein with unclear physiological function [41]. The junction between the two proteins in the precursor conforms to the consensus sequence for cleavage by proprotein convertases residing in the trans Golgi network and/or secretory vesicles [42]. Indeed, pulse-chase and subcellular fractionation experiments [25],

Gene organization

The gene for the human α1-microglobulin/bikunin precursor (AMBP) is about 20 kb long and consists of 10 exons and 9 introns [46], [47]; the first 6 exons code for α1-microglobulin, exons 8 and 9 for each Kunitz domain and exons 7 and 10 for the N- and C-terminal ends of bikunin, respectively. The 5′-region lacks a typical TATA box, but such a sequence occurs in intron 6, which is significantly larger than the other introns and separates the sequences for α1-microglobulin and bikunin. Intron 6

Expression

The transcription of the AMBP gene is regulated by a weak ubiquitous promoter and a strong, distant, liver-specific enhancer [49]. The enhancer region contains a cluster of nine binding sites for hepatocyte-enriched transcription factors, and the corresponding factors have been shown to act by both stimulating and suppressing transcription [50]. The expression of bikunin varies during the course of development: analysis of α1-microglobulin/bikunin mRNA in mouse, rat and pig fetuses revealed

Tissue distribution

Detection of bikunin mRNA in different tissues by extraction and filter hybridization has indicated that the protein is expressed only in the liver [52]. However, with a more sensitive technique — RT-PCR — the kidneys, intestine, stomach and pancreas have also been shown to express this RNA [63]. Immunochemically, bikunin has been detected in many different organs and tissues such as the liver [71], kidney [72], skin, gall bladder, bronchial mucus, cerebrum, cerebellum, testis [71], brain [73],

Plasma clearance

The plasma half-life of radiolabelled bikunin injected into humans, rats and mice is in the range of 4–30 min [7], [77], [78]. In an early study of patients suffering from various nephropathies, a clear correlation between the bikunin and creatinine concentrations in the plasma was found [79], implying that the kidneys are a major site of uptake of the protein. The same conclusion was later drawn from experiments in which rats and mice were intravenously injected with 125I-labelled bikunin [7],

Proteinase inhibitor

Bikunin has been shown to inhibit a large number of proteinases such as trypsin [80], chymotrypsin [81], granulocyte elastase [82], [83], plasmin [84], [85], cathepsin G and acrosin [86]; the reported Ki values are in the range 10−7–10−10 M, that for trypsin being the lowest [87], [88]. However, bikunin binds these enzymes less avidly than other, more abundant proteinase inhibitors in plasma, making the physiological function of bikunin unclear [87]. The amino acid residue in the binding site

Conclusions and directions for future research

The physiological role of bikunin is still unknown, but the fact that so far no patients have been found with low plasma levels indicates that this protein is essential for life. Some of the in vitro properties of bikunin that might be of physiological relevance are listed in Table 1. The finding that bikunin inhibits cell surface plasmin as well as LPS-induced stimulation of neutrophils, indicates that the protein is part of the inflammatory process. The most direct way of testing this

Acknowledgements

We thank J.-P. Salier, I. Björk and Ö. Zetterqvist for critical comments on the manuscript and B. Villoutreix for preparing Fig. 2.

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