Proteolysis in the Interstitium | 4 | Proteolysis in the Interstitial

ABSTRACT

There is compelling evidence to suggest that there are a number of proteases in the interstitial space: either secreted proteases or zymogens, membrane-bound proteases, or shed membrane proteases. A select number of these are listed in Table 4.1. Some of these enzymes are discussed in the following chapters, while in the current chapter we focus on a disintegrin and metalloproteinase (ADAM) and a disintegrin and metalloprotease with thrombospondin motif (ADAMTS). Those readers searching for a comprehensive discussion of proteolytic enzymes, including those suggested to be present in the interstitial space, may consult the recent encyclopedic work edited by Neil Rawlings and Guy Salvesen. ¹ In addition to the proteases discussed in the current work, there are a number of proteases expressed in the interstitial space during development and in tumors that are not normal constituents of the interstitial space. Many of the proteases suggested to be present in the interstitial space have not been physically isolated from a biological fluid or tissue but have rather been identified by cDNA technology. An example is provided by some members of the ADAM family described in Section 4.2. Table 4.1 Proteolytic Activities Described in the Interstitial Space

Enzyme	Comment
ADAM proteases	ADAM proteases are a group of metallproteinases ¹ ^, ² composed of a disintegrin domain and a metalloprotease domain. ¹ ADAM proteases are members of the adamlysin family in the metzincin superfamily, which also includes MMPs. ³ ^– The disintegrin domain functions in binding to integrins on the cell membrane, possibly positioning the protease to act as a sheddase. ⁴ Not all of the members of the ADAM family have proteolytic activity, suggesting the importance of protein–ligand interactions in function. ⁵ ADAM proteases have a major role in development ⁶ and are of interest in oncology research. ⁷ The function of ADAM protease as a sheddase is of importance in inflammation, as shown by the release of TNF-a by ADAM-17. ⁸ See Section 4.2 for more detail.
ADAMTS (a disintegrin and metalloprotease with thrombospondin type-1 motif repeats)	ADAMTS proteases are a group of metalloproteinases consisting of an ADAM domain (see above) and thrombospondin motif repeats. ⁹ ^– ¹¹ Unlike the ADAM proteases, which are membrane bound, the ADAMTS proteases are soluble proteins that are processed and secreted in a furin-mediated pathway. ¹² Early work suggested that the thrombospondin domains in ADAMTS proteases enabled binding to the ECM. ¹³ ADAMTS proteases are best known for their degradation of proteoglycans, such as aggrecan, ¹⁴ and collagen processing. ¹⁵ One ADAMTS protease, ADAMTS ¹³ , is involved in the processing of von Willebrand factor in endothelial cells. ¹⁶ See Section 4.3 for more detail.
Chymase	Chymase is a chymotrypsin-like serine protease that is a product of mast cells. ¹⁷ ^, ¹⁸ More specifically, chymase arising from mast cell degranulation in the arterial intimal fluid has been reported. ¹⁹ ^– ²² Chymase has biological activity in the interstitial space, such as stimulation of angiotensin II formation, ¹⁹ degradation of HDL, ³⁰ ^, ²¹ and activation of MMPs. ²³ ^– ²⁵ Demonstrated to cleave nidogen but less effective than leucocyte elastase. ²⁶ Also shown to increase glomerular permeability by cleavage of PAR-2 receptor. ²⁷
Factor VIIa	There is no direct measurement of factor VIIa in interstitial fluid, but indirect evidence supports the presence of this protease. ²⁸ ^, ²⁹ It is suggested that the formation of the thrombin in the interstitium occurs via the tissue factor pathway, which would require the conversion of factor VII to factor VIIa. ³⁰ ^, ³¹ Perivascular tissue factor binds factor VIIa. ³² See Chapter 8 for more detail.
Factor Xa	There is no direct measurement of factor Xa in interstitial fluid, but indirect evidence supports the presence of this protease. ³³ ^, ³⁴ Factor Xa can activate cells via cleavage of the PAR-2 receptor. ³⁵ See Chapter 8 for more detail.
Hepsin	A hepatic enzyme of unclear function. ³⁶ There are studies with in vitro substrates, but in vivo substrates have not been described. Hepsin is a type II transmembrane serine protease (TTSP), ³⁷ as is matripase. ³⁸ ^, ³⁹ Hepsin has been shown to activate factor VII. ⁴⁰ Hepsin is involved in malignancy and is proposed as a therapeutic target. ⁴¹ ^– ⁴⁵
Hyaluronan-binding serine protease	Hyaluronan-binding serine protease (HABP) was isolated from human plasma by affinity chromatography on hyaluronan conjugated to agarose. ⁴⁶ Analysis of amino acid sequence derived from cDNA showed homology to hepatocyte growth factor activator. Further analysis of the gene for HABP showed some homology with coagulation factor XII, tPA, and urokinase. ⁴⁷ HABP has been shown to undergo autocatalytic activation, ⁴⁸ ^, ⁴⁹ which was stimulated by the presence of either positively charged poly-lysine or negatively charged heparin. ⁴⁸ Heparan sulfate and chondroitin sulfate have also been shown to stimulate the action of hyaluronan-binding serine protease on kininogens, forming kinins. ⁴⁹ While a substrate in plasma has not been clearly identified, ⁵⁰ hyaluronan-binding serine protease has been shown to upregulate ERK1/2 and P13K/Akt, signaling pathways in fibroblasts and stimulating cell proliferation and migration. ⁵¹ The conditioned media from HABP-treated fibroblasts had a growth-stimulating effect on quiescent fibroblasts.
Insulin-like growth factor binding protein-3 protease (IGFBP-3 protease)	IGFBP-3 protease is described more as an activity ⁵² ^, ⁵³ than as a discrete molecular entity and the activity may be more a reflection of the susceptibility of IGFBP-3 to proteolysis by a wide variety of enzymes. ⁵⁴ ^– ⁶³ There is evidence to suggest that there is little proteolysis of IGFBP-3 in “normal” serum, but there is marked increase during pregancy. ⁶⁴ Proteolysis of IGFBP-3 may increase IGF-1 bioavailability. ⁵⁸ ^, ⁶⁵ Proteolysis of IGFBP-3 occurs to a higher extent in interstitial fluid. ⁶⁶ ^– ⁶⁸
Kallikrein-related peptidases ^a	A family of regulatory proteases related by structural homology and not biological function ⁶⁹ ^, ⁷⁰ (see Chapter 6).
Mastin	Mastin is a soluble tryptase-like enzyme ⁷¹ secreted by canine mast cells. ⁷² Mastin has a tryptic-like specificity with a preference for cleavage of arginine-containing peptide bonds.
Matriptase	Matriptase (MT-SP1, epitin, SNC19, TADG-15) was identified as a matrix-degrading serine protease in tumor cells. ⁷³ Subsequent studies ⁷⁴ demonstrated that matriptase was expressed on epithelial cells as a membrane-bound protease that could activate hepatocyte growth factor and urokinase plasminogen activator. Matriptase is inhibited by hepatocyte growth factor activator inhibitor 1 (HAI-1) ⁷⁵ and antithrombin. ⁷⁶ ^, ⁷⁷ Matriptase can be detected by reaction with a biotinylated peptide chloromethyl ketone. ⁷⁸ Matriptase occurs as a surface-bound zymogen that undergoes autocatalytic transactivation. ⁷⁹ Matriptase is considered to part of a proteolytic cascade involving the activation of prostasin necessary for functionality of the stratum corneum barrier function. ⁸⁰
Matrix metalloproteinase	MMPs (matrixins) ⁸¹ ^– ⁸³ are a group of proteolytic enzymes that are members of the metzincin superfamily ⁸⁴ ^, ⁸⁵ and involved in the degradation of ECM components. ⁸⁶ MMPs vary considerably in structure and size but have a common mechanism involving a cysteine residue and a metal ion, usually zinc. MMPs are synthesized as precursor or zymogen forms that require activation. ⁸⁷ ^– ⁹⁰ Glycosaminoglycans may modulate MMP action. ⁹¹ MMPs are discussed in detail in Chapter 7.
Meprin metalloproteases	Meprin A (meprin α) and meprin B (meprin β) are zinc metalloproteinases, which are astacins ⁹² in the metzincin superfamily. ⁸⁴ ^, ⁸⁵ Meprin A and Meprin B are transmembrane proteases that can be released (shed) into the interstitial space. ⁹³ ^, ⁹⁴ Meprin A may be secreted as a zymogen form. The functions of the meprin proteases are still being defined, but a number of potential substrates ⁹⁵ have been identified, including amyloid protein, ⁹⁶ procollagens, ⁹⁷ and IL-6. ⁹⁸
Mesotrypsin^b	Mesotrypsin has been described as a minor form of pancreatic trypsin derived from mesotrypsinogen by the action of enterokinase. ⁹⁹ , ¹⁰⁰ As with trypsin IV, mesotrypsin is resistant to inactivation by protein protease inhibitors. ⁹⁹ ^, ¹⁰¹ It has been suggested that mesotrypsin is important for the degradation of trypsin inhibitors such as soybean trypsin inhibitor, permitting the digestion of food rich in natural trypsin inhibitors. ¹⁰² Mesotrypsin has been described in the upper epidermis as an enzyme responsible for the activation of epidermal kallikrein-like peptidases and for the degradation of lymphoepithelial Kazal-type-related inhibitor (LEKTI1), an inhibitor of the kallikrein-like peptidases. ¹⁰³ As with the pancreatic proenzyme, epidermal mesotrypsinogen is activated by enterokinase, also found in the epidermis. These observations support a role for mesotrypsin in the desquamation process.
Neutrophil elastase	Neutrophil elastase has a chymotrypsin-like specificity and degrades elastin and collagen in the interstitial space. ¹⁰⁴ ^, ¹⁰⁵ Not to be confused with macrophage elastase (MMP-12; Chapter 8). The degradation of pulmonary elastin by neutrophil elastase is inhibited by hyaluronan. ¹⁰⁶ Neutrophil elastase has been suggested to be important for the development of interstitial edema during pericardial inflammation. ¹⁰⁷ Cleavage of E-cadherin by neutrophil elastase is suggested to result in loss of cell–cell contacts and adherens junctions in the development of experimental pancreatis. ¹⁰⁸
Neutrophil protease 3	A protease contained in neutrophils and expressed in the interstitial space after migration of the neutrophils from the vascular space. ¹⁰⁹ ^, ¹¹⁰ Neutrophil protease 3, along with other neutrophil proteases such as neutrophil elastase, is synthesized as a zymogen, processed to a mature enzyme by dipeptidyl peptidase I, ¹¹¹ and stored in azurophil granules. ¹¹⁰ A small amount of activated neutrophil protease 3 is expressed on the surface of resting neutrophils. ¹¹² Neutrophil protease 3 cleaves the PAR-1 receptor at sites separate from the thrombin cleavage site. ¹¹³ However, the cleavage of PAR-1 receptor by neutrophil proteinase-3 does activate MAP-kinase. Antibodies against neutrophil protease 3 are observed in antineutrophil cytoplasmic antigens. ¹¹⁴ ^, ¹¹⁵ The surface presentation of neutrophil protease 3 is important for its role as an antineutrophil cytoplasmic antigen. ⁹² Neutrophil migration from the vascular space into the interstitial space is critical for the normal immune response, ¹¹⁶ and the expression of neutrophil proteinase 3 is critical for the defense response in the interstitial space. ¹¹⁷ ^, ¹¹⁸
Plasma kallikrein	The presence of plasma kallikrein in the interstitial space can be inferred from the presence of bradykinin and other kinin products released by plasma kallikrein from high-molecular-weight kininogen. ¹¹⁹ ^– ¹²² Plasma prekallikrein and high-molecular-weight kininogen have been found in cerebrospinal fluid. ¹²³ Plasma kallikrein activates vascular smooth muscle cells by action on PAR receptors. ¹²⁴ There is more extensive information on plasma kallikrein in Chapter 6.
Plasmin	A serine protease derived from plasminogen that has a specificity for the cleavage of peptide bonds where the carboxyl group is contributed by lysine. ¹²⁵ Plasminogen is found in plasma and interstitial fluid; ¹²⁶ ^, ¹²⁷ plasmin can be found in serum, ¹²⁸ , ¹²⁹ urine, ¹³⁰ and synovial fluid. ¹³¹ ^– ¹³³ Plasmin is rapidly inhibited by α₂-antiplasmin, α₂-macroglobulin, and α₁-antitrypsin. ¹³⁴ ^– ¹³⁷ Cleavage of plasmin/plasminogen by a variety of proteolytic enzymes gives rise to angiostatin. ¹³⁸ ^– ¹⁴³ While best recognized for fibrinolytic activity, plasmin can directly interact with cells within the interstitium ¹⁴⁴ ^– ¹⁴⁶ and regulates the ECM and function. ¹⁴⁷ ^– ¹⁵² See Chapter 5 for more detail.
Reelin	A large glycoprotein serine protease ¹⁵³ , ¹⁵⁴ associated with the ECM that is important for nervous system development and the regulation of synaptic transmission in the adult brain. ¹⁵⁵ Reelin was identified as a product of the reeler gene, ¹⁵⁶ which is a protein component of the ECM during early cortical development ¹⁵⁷
Thrombin	A serine protease derived from prothrombin ¹⁵⁸ ^, ¹⁵⁹ that has specificity for the cleavage of peptide bonds where the carboxyl group is contributed by basic amino acids (arginine and lysine). ¹⁶⁰ ^, ¹⁶¹ Thrombin is important in the development of interstitial fibrosis. ¹⁶² ^– ¹⁶⁴ While thrombin can form fibrin in the interstitial space, ¹⁶⁵ the majority of interest is directed toward the interaction of thrombin with cells. ¹⁶² ^, ¹⁶³ ^, ¹⁶⁶ Thrombin may have a direct role in ECM degradation through the degradation of fibronectin ¹⁶⁷ and proteoglycans ¹⁶⁸ but also activates MMPs. ¹⁶⁹ ^, ¹⁷⁰ There are data to suggest that antithrombin can inactivate thrombin in the interstitial space ¹⁷¹ but there is no direct evidence for the formation of a thrombin–antithrombin complex. Thrombin can also activate hepatocyte growth factor activator. ¹⁷² See Chapter 8 for more detail.
Tissue kallikrein (KLK-1)^b	A tryptic-like serine protease best known for the formation of kinins from kininogens. ¹⁷³ It is suggested that tissue kallikrein is released into the interstitial space during inflammation ²² ^, ¹⁷⁴ or is secondary to tissue damage ¹⁷⁵ ^, ¹⁷⁶ (see Chapter 6).
Tissue plasminogen activator (tPA)	tPA is a specific activator of plasminogen that is found in a variety of tissues, ¹⁷⁷ ^, ¹⁷⁸ with some emphasis on nervous tissue ¹⁷⁹ ^, ¹⁸⁰ and eye. ¹⁸¹ There is major interest in tPA expression in endothelial cells. ¹⁸² tPA is thought to bind to endothelial cell surfaces following secretion, with such binding important for fibrinolytic activity. ¹⁸³ tPA is present in interstitial fluid ¹⁸⁴ ^, ¹⁸⁵ but is rapidly inactivated by PAI-1. ¹⁸⁶ tPA is also found on macrophage surfaces. ¹²⁸ tPA is active in a bound phase but poorly active in free solution. ¹⁸⁷ ^, ¹⁸⁸ The formation of plasmin on the melanoma cell surface, mediated by tPA activation of plasminogen bound on the cell surface, is considered important for invasiveness. ¹⁸⁹
Tryptase	Tryptases are tryptic-like serine proteases that are products of mass cells. ¹⁸ ^, ¹⁹⁰ There are several isoforms of tryptase ¹⁹¹ arising from variation in the tetramer structure of this protein. ¹⁹² , ¹⁹³ Cleavage of PAR-2 on epithelial cells by tryptase may be of importance in the interstitium. ¹⁹⁴ , ¹⁹⁵ Tryptase activates TGF-β in airway smooth muscle cells in a PAR2-independent mechanism. ¹⁹⁶ Heparin and other macropolyanions are involved in the storage and modulation of tryptase activity. ¹⁹⁷ ^– ²⁰¹ Human tryptase loses activity via a process described as spontaneous inactivation; the process of spontaneous inactivation is slowed/reversed by sulfated polyanions such as heparin or dextran sulfate. ²⁰² ^– ²⁰⁵
Trypsin IV^b	Trypsin IV is an extrapancreatic isoform of trypsin found in the brain and is derived from trypsinogen IV, which may be a splice variant of mesotrypsinogen. ²⁰⁶ ^, ²⁰⁷ Trypsin IV may also be secreted by other extrahepatic sources, including certain epithelial cells. ²⁰⁸ Trypsin IV differs from classic pancreatic trypsin in being resistant to inhibition by protein trypsin inhibitors. ²⁰⁹ Trypsin IV has the ability to activate PAR receptors and may have a role in inflammation. ²¹⁰
Urokinase plasminogen activator (urokinase)	Urokinase plasminogen activator (uPA) is an enzyme synthesized in the kidney. ²¹¹ ^, ²¹² In addition to proteolysis, urokinase acts through binding to a specific cell surface receptor (urokinase plasminogen activator receptor; uPAR). ²¹³ Urokinase and uPAR together with plasminogen activator inhibitor 1 (PAI-1) are important in inflammation ¹²⁷ and VEGF-stimulated angiogenesis. ²¹⁴ uPA is important in the programmed degradation of the ECM during development. ²¹⁵ uPA is distinct from tissue plasminogen activator (tPA). ²¹⁶ ^, ²¹⁷ See Chapter 5 for more detail.

Note: These proteins are not unique to the interstitial space but are found in other fluid compartments, including the vascular bed. See Chapter 1 for a discussion of the origin of proteins in the interstitium. a

There are some issues with the nomenclature in this area and there are studies where mesotrypsin and trypsin IV are considered interchangeable. Trypsin IV and mesotrypsin do appear to be different enzymes but share some unusual properties, such as the resistance to inactivation by protein protease inhibitors.