Mini-reviewMammalian legumain – A lysosomal cysteine protease with extracellular functions?
Introduction
The cysteine protease legumain (EC 3.4.22.34) is ubiquitously expressed in mammals with highest expression in kidneys, spleen, liver, placenta and testis [1]. Legumain displays specificity towards asparagine peptide bonds, thus synonymously termed asparaginyl endopeptidase (AEP) [1]. Later, it was shown that legumain acquires caspase-like properties at low pH (<5) by cleaving after aspartic acid [2]. In cells, legumain is primarily localized to the acidic lysosomal compartments, where activation of the protease takes place. However, legumain is shown to be extensively secreted from various cell types and reported to appear extracellularly, although only the proform has been detected [3,4]. Legumain has been found in extracellular fluids such as serum, plasma and cerebrospinal fluid (CSF) [[5], [6], [7]], and is suggested to have multiple functions both intra- and extracellularly (reviewed in Ref. [8]). In addition, legumain has been found in exosomes [9,10]. Legumain deficient (LGMN−/−) mice are born with no distinct anatomical or morphological abnormalities [11]. However, enlarged lysosomes and deficient processing of lysosomal cathepsins and toll-like receptor 9, kidney failure, and characteristic features of hemophagocytic syndrome are observed [12,13].
In vitro differentiation of human primary monocytes indicates that M1 macrophages are an important source of secreted legumain, which is measureable in plasma [7]. It has been suggested that polyubiqutinylation of prolegumain or reduced acidification of the lysosomes promote extracellular secretion, which may regulate whether legumain is retained intracellularly or secreted to the environment [14,15]. Since posttranslational polyubiquitinylation occurs in the cytosol, lysosomal prolegumain is not an obvious substrate. However, the presence of legumain in the cytosol has been described in cancer cells [16,17] and thus, such modification of legumain is possible [14]. In addition to being extensively secreted, legumain has also been reported to be present in the nucleus of colorectal cancer cells and in activated Th1 lymphocytes [17,18], and nuclear transport seems to be dependent on posttranslational glycosylation [16]. In this review we will focus on extracellular legumain and explore the hypothesis that this protein is not only functioning as a lysosomal enzyme but has additional important extracellular functions.
Legumain is synthesized as a glycosylated 56 kDa proform (prolegumain) which is auto-catalytically activated at acidic pH and reducing conditions to a 46 kDa intermediate that is further processed to a 36 kDa mature and a newly discovered 25 kDa active forms [16,19,20]. Endogenous inhibitors of legumain are the family 2 cystatins including cystatin C, E/M and F, with cystatin E/M being the most potent inhibitor [21]. Two molecular forms of cystatin E/M are expressed and secreted, a 14 kDa unglycosylated and a 17 kDa glycosylated form [22]. We have previously shown that secreted prolegumain can be internalized and subsequently processed and activated intracellularly [4]. Similarly, secreted cystatin E/M can be internalized and is able to inhibit intracellular legumain [4].
Both legumain and cystatin E/M are endogenously glycosylated, and such posttranslational modification plays an important role in protein trafficking and function [23]. In general, initiation of N-linked glycosylation occurs in the rough endoplasmic reticulum (ER), and involves attachments of high-mannose oligosaccharides to selected asparagine residues in the polypeptide [24]. During transport through ER and Golgi, the linked oligosaccharides are further modified to the formation of three main types of N-linked oligosaccharide structures (i.e. high mannose, hybrid or complex) [25]. Two N-linked glycosylation sites (N91 and N167) have been confirmed in prolegumain, although the protease has four potential N-linked sites [26]. Recently, we showed that the carbohydrates on legumain are of the hybrid or high mannose type, whereas cystatin E/M has complex mannose-linked carbohydrates [16]. N-glycosylation of prolegumain is necessary for cellular internalization and correct processing to active forms, whereas cystatin E/M is independent of the glycosylation status for inhibition of legumain [16]. In addition to function as legumain inhibitors, both cystatin C and E/M are substrates of legumain [[27], [28], [29]] (Table 1). Furthermore, inhibition of posttranslational glycosylation facilitates cell secretion of prolegumain [16].
The prevailing theory for intracellular transport of newly synthesized legumain is through the trans-Golgi network and further into lysosomes through endosomes (Fig. 1; reviewed in Refs. [8,30]). It is assumed that glycosylation is important for transport to the lysosomes, the main compartment for prolegumain autoactivation [15]. A mannose-6-phosphate (M6P) motif has been reported on the N-terminus of legumain [31] and could target legumain to the vesicular pathway depending on the interaction with different M6P-receptors. The cation-dependent M6P-receptor is important for routing prolegumain and precursors of other lysosomal enzymes from Golgi to the late endosomes, while the cation-independent M6P-receptor on the cell surface facilitates re-capturing of secreted proenzymes, followed by clathrin-mediated endocytosis and subsequent trafficking to the lysosomes [32,33]. We have recently shown that internalization of legumain is not through clathrin-mediated endocytosis since blocking of this endocytosis pathway did not inhibit legumain internalization [16]. Thus, the exact internalization mechanism of legumain is still unknown and needs further investigations. However, rerouting from classical transport pathways is often associated with pathological conditions [34]. A previous review addressed myths and common questions regarding localization and trafficking of endo-lysosomal cysteine proteases [30]. Nevertheless, the regulation and mechanisms of legumain trafficking in or out of cells is still poorly understood. Similarly to legumain, both cystatin C and E/M are internalized [4,35] and thus, the interplay between legumain and its inhibitors appears to take place both intra- and extracellularly.
Secreted prolegumain could be activated in acidic extracellular microenvironments during pathological or inflammatory conditions. Prolegumain is stable in neutral pH, whereas mature legumain is unstable and rapidly inactivated at pH > 6 [1,20]. Stabilization of the proenzyme is postulated to be mediated by the positively charged C-terminal domain located on top of the protease catalytic domain [27,36]. Legumain is stabilized in the extracellular environment by interactions with integrins, glycosaminoglycans (GAGs) or cystatins forming amyloid fibril fragments (Fig. 1; [29,36,37]) and such interactions might increase the half-life of the protease. Since the nature of cystatin inhibition is reversible, amyloid fibrils could act as a legumain storage [29]. The legumain structure contains a RGD motif which allows binding to integrin receptors on the cell surface [38]. Legumain binding to the αVβ3 integrin results in increased stability, catalytic activity and shift in the pH optimum from pH 5.5 to 6.0 [36]. Similarly, we have previously shown that extracellular matrix (ECM) components, such as the GAGs chondroitin 4-sulfate (C4S) and heparan sulfate (HS), accelerate prolegumain autoactivation in a pH-, concentration- and time-dependent manner [37] and the carbohydrates on legumain seem to play a key role in the interaction between prolegumain and GAGs [16]. Thus, the interplay between legumain and components in the extracellular milieu is of great interest to understand the extracellular roles of this protease.
Legumain has been postulated to have various activities, i.e. asparaginyl endopeptidase (AEP), asparaginyl carboxypeptidase and ligase activities (reviewed in Ref. [8]). The suggested extracellular AEP-substrates are presented in Table 1. Below, we will focus on extracellular legumain (AEP) in pathology, as legumain has been shown to be involved in inflammation, atherosclerosis, bone homeostasis and osteoporosis, cancer and neurodegenerative diseases. In addition, legumain has been suggested as a biomarker of idiopathic pulmonary fibrosis [45], liver fibrosis [46] and pancreatitis [47].
Section snippets
Extracellular legumain in atherosclerosis
Atherosclerosis is a major cause of cardiovascular disease and responsible for high mortality and morbidity world-wide. In the arteries, lipids and immune cells are central players, driving the persistent inflammatory process which is characteristic for atherosclerosis [48,49]. During inflammation, pro-inflammatory mediators are secreted, promote lesion formation and are responsible for plaque destabilization. Proteases, especially matrix metalloproteases (MMPs), are implicated in
Extracellular legumain in bone homeostasis and osteoporosis
Bone is a complex tissue composed of bone forming (osteoblasts, OBs) and bone resorptive (osteoclasts, OCs) cells, as well as solid extracellular matrix. It is well known that bone cells secrete proteins with autocrine, paracrine or endocrine functions. Legumain has been identified in the secretome of human bone marrow skeletal (mesenchymal) stem cells (hBMSC) during ex vivo OB differentiation [69]. Human BMSCs are multipotent non-hematopoietic stromal cells that can differentiate into OBs or
Extracellular legumain in cancer
Several studies have linked legumain to the development, progression and severity of cancer. A meta-analysis has shown that legumain is overexpressed in cancer compared with normal tissue and was higher in late stages (III-IV) than in early stages (I-II) of disease [76]. Moreover, legumain overexpression was correlated with poor prognosis and clinical stage. Although being primarily a lysosomal protease, legumain is secreted by cancer cells to the extracellular space. Tumours generate acidic
Extracellular legumain in neurodegenerative diseases
Neurodegenerative diseases (NDG) share a common feature in accumulation of proteins that are processed aberrantly and misfolded. These proteins form neurotoxic aggregates that cause diseases such as Alzheimer's disease (AD) and Parkinson's disease. Clearance of such proteins is important for preventing development of NDG [94], and autophagy or protein degradation in lysosomes are crucial. Legumain has been shown to be involved in autophagy [95] and the presence in lysosomes contributes to
Perspectives
Further studies are required to understand the molecular mechanisms, regulation and cellular interplay of secreted legumain and cystatins, and whether legumain can be used as a biomarker, pharmacological target or be utilized for drug targeting in the treatment of various diseases. Of special interest is to unequivocally establish whether legumain exists as an active enzyme (AEP) extracellularly and to unravel its local substrates. Furthermore, possible legumain functions not dependent on the
Conflict of interest
None.
Author's contribution
All authors have contributed equally and approved the final manuscript.
Acknowledgements
This work is supported by the University of Oslo.
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