The early history of intracellular cysteine proteinase inhibitors

Ari Rinne

University of Tromso, Department of Pathology, NO-9037 Tromso, Norway

TABLE OF CONTENTS

1. Abstract

2. Introduction

2. Introduction

3. A short history of inhibitors and corrections to Turk et al

4. Acknowledgements

5. References

1. ABSTRACT

Scientists at the beginning of their careers often rely on the information obtained from review articles. Furthermore, the review articles are commonly used by sponsors in making their funding decisions of scientific projects. Thus it is very important to give a balanced view of the past as well as the present of the field discussed in those papers. In the present letter I wish to complement a review article previously published in Frontiers in Bioscience.

2. INTRODUCTION

A recent review calls for critical discussion and an analysis of the basic findings in the field of cysteine proteinase inhibitors. In an article of Turk et al. the most basic findings in the field were neglected (1). As a result of that, the much later article is referred to as the pioneering study in the field (2). I have worked with cysteine proteinase inhibitors almost from the very beginning of the history of these proteins. Thus, it is my duty for the future generations of scientists to share my views regarding the first steps of the inhibitor studies and fill some gaps in the information given in Turk et al. (1).

Objective and ethical information in review articles has recently become even more important as the funding of science in the member states of the EU has largely shifted from national to EU-based sources. It is well known that the vast number of the applications and the limited time allotted for their processing forces the board members of the EU's scientific funds to rely on review articles instead of original literature. In addition, young scientists at the beginning of their careers often rely on review articles. Therefore, it is important that the information in these review articles is as reliable as possible, and both the writer and the publisher must feel their responsibility.

3. A SHORT HISTORY OF INHIBITORS AND CORRECTIONS TO TURK ET AL

The Japanese scientists Tokuda, Tokaji, Udaka and Hayashi were the first to find intracellular mammalian cysteine proteinase inhibitors in the 1960's. They purified, characterized and crystallized the inhibitor from rabbit skin. They also studied the role of the inhibitor in the Arthus inflammatory reaction (3, 4, 5, 6).

My own experimental knowledge of these inhibitors stems from the year 1975 when I joined the group of Dr. Mikko Järvinen in the Department of Pathology, University of Oulu, Finland. Järvinen had recently isolated BANA hydrolase from the rat skin (7). This enzyme is nowadays known as cathepsin H (8). During this work he found that dilution of the rat skin extract greatly increased the specific activity of the enzyme in the extract. This finding led him to search for a reversible inhibitor(s) in the extract. During the course of that work Järvinen developed the papain-Sepharose affinity purification method for the inhibitors, which, with minor modifications, is still used all over the world (9). Two distinct inhibitors, with molecular weights of about 74000 (I₁), and 13000 (I₂) were purified and characterized. In 1976 the low-molecular inhibitor was also demonstrated in the human skin (10).

Already in 1976 we had a specific antibody of rat I₂ which I began to use in pathological anatomy in rat tissues (11). The purification, characterization and preparation of the antibody of the small molecular inhibitor from human epidermis was published in 1978 (12), and the purification of large molecular inhibitor (I₁) from human serum in 1979 (13). Later I₁ was shown to be the kininogen (14, 15).

We immediately started an immunological and physicochemical screening of the inhibitors in human and rat tissues. These screening studies revealed that there are two different small-molecular inhibitors in the lymphatic tissue (16, 17). They had different isoelectric pHs, and the more acidic inhibitor, which was identical to that in epidermis (I₂), was called Acid Cysteine Proteinase Inhibitor ACPI (later named Cystatin A) and that with neutral isoelectric pH Neutral Cysteine Proteinase Inhibitor NCPI (later named Cystatin B) (18). Very soon it was shown the cleary different topology of these inhibitors in various tissues, e.g. in blood cells (18).

In the review article Turk et al. (1) the history of cysteine proteinase inhibitors starts from the chicken egg white inhibitor purified by Fossum and Whitaker in 1968 (19) and the first intracellular inhibitor from the work of Kopitar et al 1978 (2). The absence of the basic works of Udaka and Hayashi (1965), Järvinen (1975) and others can seemingly be explained by saying: "However, it is impossible to include most of the references due to their enormous number" (1). In my opinion, when writing a review article, it is important to bring forward a balanced picture of the development of the field. I agree that some kind of excluding of references is necessary but it must not result in the hiding of innovative key articles, lack of which could mislead the reader.

Further, the authors of the review write: "The first intracellular protein inhibitor of papain, cathepsin B and H was isolated and partially characterized from pig leucocytes and spleen" (1). The study of Kopitar (1978) contains valuable and independent information, but based on my previous discussion it cannot be the first innovative publication on intracellular cysteine proteinase inhibitors. The writers of the review (1) move forward the time invention of the mammalian inhibitors by almost 20 years. The early studies by Udaka and Hayashi (1965) from Japan and Järvinen (1975) from Finland are distinct innovations on which a great number of the later studies have been founded or at least got their basic ideas from. The information in those papers is still in most respects concrete and up to date. They can still be recommended as basic reading for young scientists working with cysteine proteinase inhibitors.

4. ACKNOWLEDGEMENTS

I wish to thank Mrs. Soili Huhta for excellent secretarial work.

5. REFERENCES

1. V Turk, V Stoka, D Turk: Cystatins: Biochemical and structural properties, and medical relevance. Front Biosci 5406-5420 (2008)
doi:10.2741/3089

2. M Kopitar, J Brzin, T Zvonar, P Locnicar, L Kregar, V Turk: Inhibition studies of an intracellular inhibitor on thiol proteinases. FEBS Lett 91, 355-359 (1978)
doi:10.1016/0014-5793(78)81209-5

3. A Tokuda, H Hayashi, K Matsuba: Biochemical study of cellular antigen-antibody reaction in tissue culture. II. Release of a protease inhibitor. J Exp Med 112, 249-255 (1960)
doi:10.1084/jem.112.2.249

4. K Udaka, H Hayashi: Molecular-weight determination of a protease inhibitor from rabbit skin with healing inflammation. Biochim Biophys Acta 104, 600-603 (1965)

5. G Tokaji: The chemical pathology of thermal injury, with special reference to burns SH-dependent protease and its inhibitor. Kumamoto Med J 24, 68-86 (1971)

6. H Hayashi: The intracellular neutral SH-dependent protease associated with inflammatory reactions. Review. Int Rev Cytol 40, 101-151 (1975)
doi:10.1016/S0074-7696(08)60952-3

7. M Järvinen, VK Hopsu-Havu: α-N-benzoylarginine-2-naphthylamide hydrolase (Cathepsin B1?) from rat skin. II. Purification of the enzyme and demonstration of two inhibitors in the skin. Acta Chem Scand B 29, 772-780 (1975)
doi:10.3891/acta.chem.scand.29b-0772

8. H Kirschke, J Langner, B Wiederanders, S Ansorge, P Bohley, H Hanson: Cathepsin H: an endoaminopeptidase from rat liver lysosomes. Acta Biol Med Ger 36, 185-199 (1977)

9. M Järvinen: Purification and properties of two protease inhibitors from the skin inhibiting papain and other SH-proteases. Acta Chem Scand (B) 30, 933-940 (1976)
doi:10.3891/acta.chem.scand.30b-0933

10. JE Fräki: Human skin proteases. Separation and characterization of two acid proteases resembling cathepsin B1 and cathepsin D and of an inhibitor of cathepsin B1. Arch Dermatol Res 255, 317-330 (1976)

11. A Rinne, M Järvinen, O Räsänen: A protein reminiscent of the epidermal SH-protease inhibitor occurs in squamous epithelia of man and rat. Acta Histochem 63, 183-192 (1978)

12. M Järvinen: Purification and some characteristics of the human epidermal SH-protease inhibitor. J Invest Dermatol 71, 114-118 (1978)
doi:10.1111/1523-1747.ep12546165

13. M Järvinen: Purification and some characteristics of two human serum proteins inhibiting papain and other thiol proteinases. FEBS Lett 108, 461-464 (1979)
doi:10.1016/0014-5793(79)80588-8

14. I Ohkubo, K Kurachi, T Takasawa, H Shiokawa, M Sasaki: Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry 23, 5691-5697 (1984)
doi:10.1021/bi00319a005

15. W Müller-Esterl, H Fritz, W Machleidt, A Ritonja, J Brzin, M Kotnik, V Turk, J Kellermann, F Lottspeich: Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data. FEBS Lett. 182, 310-314 (1985)
doi:10.1016/0014-5793(85)80322-7

16. A Rinne, M Järvinen, J Martikainen, M Alavaikko, O Räsänen: Über das Vorkommen des epidermalen SH-protease-inhibitors im lymphatischen Gewebe. Verh Anat Ges 75, 537-538 (1981)

17. M Järvinen, A Rinne: Human spleen cysteine proteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants. Biochim Biophys Acta 9, 210-217 (1982)

18. ME Davies, AJ Barrett: Immunolocalization of human cystatins in neutrophils and lymphocytes. Histochemistry 80, 373-377 (1984)
doi:10.1007/BF00495420

19. K Fossum, JR Whitaker: Ficin and papain inhibitor from chicken egg white. Arch Biochem Biophys 125, 367-375 (1968)
doi:10.1016/0003-9861(68)90672-3

Key Words: Cystatin, Medical ethics, Review