Regulatory enzymes

The Immune-Neuroendocrine Circuitry Edited by I. Berczi and A. Szentivanyi 9 2003 Elsevier Science B.V. All rights reserved

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Regulatory Enzymes

ISTVAN BERCZI and EDRIS SABBADINI

Department of Immunology, Faculty of Medicine, The University of Manitoba, Winnepeg, Manitoba, Canada R3E OW3 ABSTRACT

Enzymes of the serine protease family induce a wide range of biological effects by activating several hormones and growth factors and by participating in enzyme cascades for inflammation, blood coagulation, complement activation, and other reactions. The action of these proteases is regulated by the endocrine system and by several inhibitors, also under endocrine control. Many inhibitors of these enzymes belong to a single family of proteins called serine protease inhibitors or serpins. This review will deal first with the components of the contact system and with the mechanisms of activation of its primary enzymes and then with the functions of other serine proteases and their inhibitors. Finally, a brief discussion about the roles of serine proteases in physiological reactions and in disease states will be presented.

1.

THE CONTACT SYSTEM

The main components of the contact system are three zymogens: Hagemann factor (HF), also referred to as coagulation factor XII (FXII), plasma pre-kallikrein (PK), and coagulation factor XI (FXI). These zymogens can be activated by partial proteolysis into enzymatically active serine proteases. In addition, a fourth primary component, high molecular weight kininogen (HMWK), acts as a co-factor in the activation of these enzymes and as a substrate for the active plasma kallikrein (K). The activation of the primary enzymes leads to activation of the intrinsic coagulation cascade, to the triggering of the complement cascade via the classical pathway, fibrinolysis via plasminogen activation, and inflammatory reactions via the activation of plasma kallikrein with the subsequent production of vasodilatatory kinins. Effects on blood pressure may also be induced by the activation of prorenin and by the hypotensive effects of kinins (Figure 1). HF is a ~-globulin of 74-80 kDa molecular weight [ 1,2] contained in plasma. The molecule can be cleaved at various points by limited proteolysis giving rise to enzymatically active factors, the main ones being c~HFa of 80 kDa and [~HFa of 28 kDa. The initial activation of the contact system is probably due to the binding of HF to negatively charged surfaces where the inactive molecules generate small amounts of enzymatically active o~HFa which activates more HF molecules. The reaction is further amplified by plasma kallikrein (K) which is activated by the two active HF products and, in turn, activates HE Of the two main activation products of HF, aHFa retains large numbers of positively charged amino acids in its heavy chain and remains attached to the negatively charged surface where the reaction was initiated. On this surface,

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Auto.activatig"-""~y HF~-~ ......~FIXI .... HF \ PK FXIa--~[coagulation l ~,~_.,...KV /

"

]C[1__ -Cimple~inl lActivati~ 1 Plasminogen ~ Pl~min

[1nnammation]

~ypotensi0n' / Abbreviations;

Ct

FXI I-IF PK F i g u r e 1. S c h e m a t i c

= = = =

[Fibr~olysis !

The first component of complement Coagulation factor XI; FXla = activated FX1; Hagemann factor; Ha = activated FH Pre-kallikrein representation

of the contact activation cascade.

o~HFa can activate FXI and trigger the intrinsic blood coagulation cascade. On the other hand, [3HFa is soluble and returns to the fluid phase. It does not participate in blood coagulation but can induce the activation of plasma PK and of C 1. Kallikrein circulates in blood as the zymogen PK, a gamma globulin of 88 kDa [2,3]. In the circulation, PK is normally bound to its co-factor high molecular weight kininogen (HMWK). Since this is rich with positively charged amino acids, it will bind to negatively charged surfaces and bring PK in contact with HF, which also binds to such cell surfaces. PK is then activated through limited proteolysis by the products of HF activation. The active K molecule has a molecular weight of 36 kD. Its main substrate is HMWK from which the nonapeptide bradykinin is generated. Besides this action on kininogen, K interacts with HF which it activates, resulting in the amplification of the contact activation cascade. It also acts on FXI which it activates into FXIa and with plasminogen which it activates into plasmin, the main enzyme of the fibrinolytic system. The last action proceeds slowly and may have only minor physiological significance. FXI is a gamma globulin of 80 kDa, [4] that in circulation is bound to HMWK and can be activated through limited proteolysis by ~HFa on a negative surface. Amino acid sequencing of FXI shows significant homologies with PK, indicating that the two proteins may have a common evolutionary origin. The main function of FXI is the proteolytic activation of factor IX which is an essential component of the intrinsic pathway of blood coagulation. Deficiencies in FXI produce a hemophilia like syndrome [5]. Activated FXI also activates HF and plasminogen. There are two major kininogens in humans and most other mammalian species, HMWK of 110 kDa and low molecular weight kininogen (LMWK) of 60 kD, [3,6] the latter being contained in plasma in concentrations 4-5 times higher than HMWK. The two molecules originate from a single gene which gives rise to two messenger RNA molecules of different lengths. HMWK is the main substrate of plasma K. Plasma K cleaves HMWK at a fast rate but cleaves LMWK slowly. On the other hand, tissue kallikrein cleaves both kininogens with equal efficiency. From the action of plasma K on kininogens, the nonapeptide bradykinin is obtained, while the action of tissue kallikrein results in the production of the decapeptide kallidin (lys-

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bradykinin). Kininogens are multifunctional molecules. Besides being the source of kinins, they can inhibit the function of several peptidases. Moreover, HMWK circulates in association with PK and FXI, which it prevents from being activated while in plasma. Because of its ability to bind to negatively charged surfaces, HMWK brings these factors in contact with HF whenever a tissue injury exposes such negatively charged surfaces. Thus, the contact system is activated only when a trauma or infection produces sufficient tissue disruption and exposes negatively charged surfaces. The kinins bradykinin and lys-bradykinin have similar action and act via the same receptors expressed on various cells but particularly on smooth muscle cells. There are three well characterized kinin receptors BK~, BK 2, and BK 3, of which the B K 2 receptor is the most abundant. The BK 2 receptor is homologous to the H 2 histamine receptor and to the neurotensin and tachykinin receptors. The effects of the two kinins consist of vasodilation, hypotension and bronchial smooth muscle contraction. Pain is also elicited either directly or via the stimulation of prostaglandin production. All these actions result in strong inflammatory effects. Moreover, the hypotensive effects, effects on electrolytes and modulatory effects on the renin-angiotensin system are thought to be important in counteracting the hypertensive effects of the renin-angiotensin system and, therefore, in the regulation of blood pressure. Kinins have a very short half life in blood and tissues. They are rapidly degraded by enzymes, collectively referred to as kininases, which limit their potentially harmful effects. Kininase I is a carboxypeptidase that accounts for most of the bradykinin destroying activity of human plasma. Kininase II was shown to be identical to the angiotensin converting enzyme (ACE). Several inhibitors of the contact system components are present in the circulation [2]. The main ones are: C1 inhibitor, which is a potent inhibitor of C1 but also inhibits HFa quite strongly; K, and FXIa, the ~Zz-plasmin inhibitor or ot2-antiplasmin, which strongly inhibits plasmin activity and more weakly HFa activity; o~2-macroglobulin, which inhibits HFa and K; antithrombin III, an inhibitor of HFa and K; and ~-antitrypsin inhibitor, an inhibitor of FXIa.

2.

TISSUE KALLIKREINS

Tissue or glandular kallikreins are a group of serine proteases that differ from plasma K in terms of enzymatic action and genes of origin. They form a family of proteins controlled by homologous genes that appear to originate from a common ancestor gene, probably via a series of gene duplications and differentiations. The best studied of these enzymes is true glandular kallikrein, usually referred to as K1, found in all mammalian species studied so far. The other members of the family, called K2, K3, etc., differ from species to species. There are three well characterized tissue K in humans, hK1, hK2, and hK3, and a fourth gene has been described. In mice 24 genes have been described and 20 in rats [7,8]. Though highly homologous, the different members of the kallikrein family act on different substrates and have different tissue distributions. Their actions are believed to be mainly local, in the sense that they are believed to take place either in the tissue itself or in the local circulation immediately after release. Most members of the tissue K family have been shown to be dependent on several hormones [2,9]. Thus, androgens increase the production of many K family enzymes in salivary glands and in the prostate, and probably in other tissues. Prolactin and growth hormone have been suggested to have similar function of promoting K family enzyme synthesis. Estrogens increase the production of K1 in the pituitary gland and in the brain. In humans, as in most other species, K1 is particularly abundant in the pancreas, in the kidney

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and salivary glands [10]. It is also found in various body fluids such as saliva, urine, plasma and bile. Kidney K1 is secreted in urine. It is believed to have a significant role in the maintenance of normal blood pressure by contributing to the regulation of blood flow in the kidneys, by helping in the control of electrolyte and water transport, and by interacting (in a manner not completely clarified) with the renin-angiotensin system. In salivary glands, K1 is contained in the granules of convoluted tubules. It can be secreted in saliva and, in smaller quantities, in plasma. It probably contributes to regulation of blood flow in the salivary glands and, at least in the rat, appears to have a major role in the maintenance of the normal functions of the mucosa associated lymphoid tissue of the gastrointestinal tract. K1 was also found to be produced in several other tissues. These include the stomach, intestine, heart, blood vessels, brain, and endocrine organs such as the pituitary, ovary, testis and the adrenals. Pituitary K1 was shown to be estrogen dependent, blocked by bromocriptine, and to be involved in the processing of prolactin and proopiomelanocortin (POMC). K1 was shown to have several effects on the immune system; therefore, several authors have described mitogenic and co-mitogenic effects of kallikrein and other serine proteases. Such mitogenic effects were observed in thymocytes [11 ], T cells and B cells [12]. Although bradykinin may also have mitogenic effects [13], the involvement of this kinin in kallikrein-induced mitogenesis is not well investigated. Moreover, several proteases, including kallikrein, were shown to be involved in immunoglobulin isotype control. The addition of kallikrein and other serine proteases to cultures of B cells stimulated with LPS and IL-4, depending on concentration, enhanced the production of IgE, IgG1, or IgG3 [14]. When injected subcutaneously, K1 suppressed cell mediated and humoral immune responses [ 15]. On the other hand, if given orally K1 favored the induction of tolerance to antigens administered orally either at the same time or shortly after K1. Human K3, also known as prostate specific antigen (PSA), is found in high concentrations in the prostate gland, from which small amounts are released into the plasma [ 16,17]. During the development of prostatic neoplasias, marked increases in plasma concentrations of PSA are observed. These increases in plasma concentration are thought to be mainly due to the tissue disruption induced by the invasive neoplasia. As a result, PSA concentration can be used as a diagnostic tool for the early detection and for the post-surgical follow up of prostatic cancer. The normal function of human K3 appears to be the induction of the liquefaction of spermatic fluid, thanks to its action on the protein semenogelin, the major component of the sperm coagulum. Human K2 is also found in significant amounts in the prostate. Its function is not clear, although it has been suggested that it may synergize with K3 in sperm liquefaction [ 16,17]. In species other than humans, only some members of the K family have clear functions. In mice, the two kallikreins y-NGF and [3-NGF form a complex with the nerve growth factor (NGF) molecule and are probably involved in its activation. Similarly, the so called epidermal growth factor binding proteins (EGF-BPs) are kallikrein family members that form complexes with and activate the EGF molecule. Mouse renin and rat tonin are also members of the K family and have hypertensive effects.

3.

OTHER SERINE PROTEASES

Other serine proteases have a variety of functions. Trypsin and chymotrypsin are serine proteases with well known digestive functions. These are not discussed in any detail here. Several serine proteases are involved in the destruction and reshaping of the extracellular matrix.

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Enzymes such as elastase, collagenase, cathepsin G can solubilize elastin and collagen in various connective tissues. Cathepsin G, elastase released by neutrophils, and chymase released by mast cells, appear to be involved in several inflammatory diseases, such as arthritis and gingivitis, in emphysema and in the invasion of tissues by malignant tumors. In the immune system, activated cytotoxic T cells and natural killer (NK) cells contain granules rich with the serine protease Granzyme B. This enzyme acts in concert with enzyme, perforin, another enzyme also contained in the granules of these cells, to induce apoptosis of target cells [18]. Mast cells contain chymase, a serine protease with neuropeptide degrading functions, capable of generating angiotensin II from angiotensin I. This would constitute an alternative pathway to the one provided by the angiotensin converting enzyme (ACE) in the renin/angiotensin system. Some not well identified serine proteases have been shown to modify or suppress immune responses. Ishizaka described a kallikrein-like factor called glycosylationenhancing factor, which induced CD4+ T cells to produce IgE-potentiating factor and to favor the production of IgE memory B cells [19]. Serine proteases from Schistosoma mansoni schistosomula were reported to enhance IgE production [20].

4.

SERPINS

Serpins are a family of more than 50 proteins [21 ] sharing similar amino acid sequences and are found in vertebrates, invertebrates, viruses and plants. There are two groups of serpins, those which inhibit serine proteases and those which do not. Proteins such as ovalbumin, angiotensinogen and thyroxine binding protein, have strong homologies with the rest of the family but are devoid of protease inhibiting activity. On the other hand, most serpins are strong inhibitors of serine protease activity. They bind in a stoichiometric (1:1) fashion to the active site of their specific target enzyme(s) and exert their inhibitory function in this way. This wide phylogenetic distribution and the variety of functions supported by these proteins suggest that they have a significant role in evolutionary fitness. The functions of several serpins in the regulation of inflammatory processes, in the coagulation cascade, in complement activation and in the fibrinolysis process have been described above. Protease nexin I (PNI), thrombin inhibitor produced by glia and found in cerebrospinal fluid, was suggested to be contained in the amyloid plaques of patients with Alzheimer disease. Aprotinin, a serpin from bovine pancreas, has been extensively studied [22]. It has been used therapeutically in situations, such as major surgical procedures and pancreatitis, in which the extensive activation of the fibrinolytic system may lead to complications. Several serpins have been obtained from hematophagous invertebrates which use them to block coagulation of blood in the parasitized species.

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