Niels Bovenschen

The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.

CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy

In diabetic nephropathy, connective tissue growth factor (CTGF) is upregulated and bone morphogenetic protein 7 (BMP-7) is downregulated. CTGF is known to inhibit BMP-4, but similar cross-talk between BMP-7 and CTGF has not been studied. In this study, it was hypothesized that CTGF acts as an inhibitor of BMP-7 signaling activity in diabetic nephropathy. Compared with diabetic wild-type CTGF(+/+) mice, diabetic CTGF(+/-) mice had approximately 50% lower CTGF mRNA and protein, less severe albuminuria, no thickening of the glomerular basement membrane, and preserved matrix metalloproteinase (MMP) activity. Although the amount of BMP-7 mRNA was similar in the kidneys of diabetic CTGF(+/+) and CTGF(+/-) mice, phosphorylation of the BMP signal transduction protein Smad1/5 and expression of the BMP target gene Id1 were lower in diabetic CTGF(+/+) mice. Moreover, renal Id1 mRNA expression correlated with albuminuria (R = -0.86) and MMP activity (R = 0.76). In normoglycemic mice, intraperitoneal injection of CTGF led to a decrease of pSmad1/5 in the renal cortex. In cultured renal glomerular and tubulointerstitial cells, CTGF diminished BMP-7 signaling activity, evidenced by lower levels of pSmad1/5, Id1 mRNA, and BMP-responsive element-luciferase activity. Co-immunoprecipitation, solid-phase binding assay, and surface plasmon resonance analysis showed that CTGF binds BMP-7 with high affinity (Kd approximately 14 nM). In conclusion, upregulation of CTGF inhibits BMP-7 signal transduction in the diabetic kidney and contributes to altered gene transcription, reduced MMP activity, glomerular basement membrane thickening, and albuminuria, all of which are hallmarks of diabetic nephropathy.

The B domain of coagulation factor VIII interacts with the asialoglycoprotein receptor

Summary.u2002 Background:u2002Coagulation factor VIII (FVIII) is a heavily glycosylated heterodimeric plasma protein that consists of a heavy (domains A1‐A2‐B) and light chain (domains A3‐C1‐C2). It has been well established that the clearance of FVIII from the circulation involves mechanisms that are sensitive to the low‐density lipoprotein receptor (LDLR) family antagonist receptor‐associated protein (RAP), including LDLR‐related protein. Because FVIII clearance in the presence of a bolus injection of RAP still occurs fairly efficient, also RAP‐independent mechanisms are likely to be involved. Objectives:u2002In the present study, we investigated the interaction of FVIII with the endocytic lectin asialoglycoprotein receptor (ASGPR) and the physiological relevance thereof. Methods and results:u2002Surface plasmon resonance studies demonstrated that FVIII dose‐dependently bound to ASGPR with high affinity (Kdu2003≈u20032u2003nm). FVIII subunits were different in that only the heavy chain displayed high‐affinity binding to ASGPR. Studies employing a FVIII variant that lacks the B domain revealed that FVIII‐ASGPR complex assembly is driven by structure elements within the B domain of the heavy chain. The FVIII heavy chain‐ASGPR interaction required calcium ions and was inhibited by soluble d‐galactose. Furthermore, deglycosylation of the FVIII heavy chain by endoglycosidase F completely abrogated the interaction with ASGPR. In clearance experiments in mice, the FVIII mean residence time was prolonged by the ASGPR‐antagonist asialo‐orosomucoid (ASOR). Conclusions:u2002We conclude that asparagine‐linked oligosaccharide structures of the FVIII B domain recognize the carbohydrate recognition domains of ASGPR and that an ASOR‐sensitive mechanism, most likely ASGPR, contributes to the catabolism of coagulation FVIII in vivo.

NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network.

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component α-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, α-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct α-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A.

Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide- and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein β-tubulin after two distinct Arg residues. Neither GrK cleavage sites in β-tubulin nor a peptide-based proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.

Orphan granzymes find a home

Summary:u2002 Cytotoxic lymphocytes are armed with granules that are released in the granule‐exocytosis pathway to kill tumor cells and virus‐infected cells. Cytotoxic granules contain the pore‐forming protein perforin and a family of structurally homologues serine proteases called granzymes. While perforin facilitates the entry of granzymes into a target cell, the latter initiate distinct apoptotic routes. Granzymes are also implicated in extracellular functions such as extracellular matrix degradation, immune regulation, and inflammation. The family of human granzymes consists of five members, of which granzyme A and B have been studied most extensively. Recently, elucidation of the specific characteristics of the other three human granzymes H, K, and M, also referred to as orphan granzymes, have started. In this review, we summarize and discuss what is currently known about the biology of the human orphan granzymes.

Macrophage Low-Density Lipoprotein Receptor-Related Protein Deficiency Enhances Atherosclerosis in ApoE/LDLR Double Knockout Mice

Objective—In vitro studies implicate that the low-density lipoprotein receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of lipoproteins. Methods and Results—We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3+ T cells were observed in lesions from macrophage specific LRP-deficient mice. Conclusions—Our data demonstrate that independent of its role in lipoprotein uptake, absence of LRP in macrophages resulted in more advanced atherosclerosis and in lesions that contained more collagen and less CD3+ T cells. In contrast to previous in vitro studies, we conclude that macrophage LRP has an atheroprotective potential and may modulate the extracellular matrix in the atherosclerotic lesions.

Proteolytic cleavage of factor VIII heavy chain is required to expose the binding-site for low-density lipoprotein receptor-related protein within the A2 domain

Summary.u2002 Background:u2002Low‐density lipoprotein receptor‐related protein (LRP) is an endocytic receptor that contributes to the clearance of coagulation factor (F) VIII from the circulation. Previously, we have demonstrated that region Glu1811‐Lys1818 within FVIII light chain constitutes an important binding region for this receptor. We have further found that FVIII light chain and intact FVIII are indistinguishable in their LRP‐binding affinities. In apparent contrast to these observations, a second LRP‐binding region has been identified within A2 domain region Arg484‐Phe509 of FVIII heavy chain. Objective:u2002In this study, we addressed the relative contribution of FVIII heavy chain in binding LRP. Methods and Results:u2002Surface plasmon resonance analysis unexpectedly showed that FVIII heavy chain poorly associated to the receptor. The binding to LRP was, however, markedly enhanced upon cleavage of the heavy chain by thrombin. The A2 domain, purified from thrombin‐activated FVIII, also showed efficient binding to LRP. Competition studies employing a recombinant antibody fragment demonstrated that region Arg484‐Phe509 mediates the enhanced LRP binding after thrombin cleavage. Conclusions:u2002We propose that LRP binding of non‐activated FVIII is mediated via the FVIII light chain while in activated FVIII both the heavy and light chain contribute to LRP binding.

Interaction between factor VIII and LDL receptor-related protein. Modulation of coagulation?

Recent reports suggest that the multifunctional receptor low-density lipoprotein receptor-related protein (LRP) may contribute to the regulation of blood coagulation by mechanisms that differ from the simple removal of protease/inhibitor complexes from the circulation. This possibility became apparent from the observation that LRP is involved in down-regulation of Tissue Factor expression at the surface of monocytes and fibroblasts. Furthermore, coagulation Factor VIII and activated Factor IX (Factor IXa) have been identified as proteins that are able to bind to LRP. In the present review, the potential contribution of LRP to the regulation of the coagulation cascade through these novel pathways is discussed, with particular reference to the interaction between LRP and coagulation Factor VIII.

Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha.

Hypoxia triggers the transcription of genes responsible for cell survival via the key player transcription factor hypoxia-inducible factor 1alpha (HIF-1α). Overexpression of this protein has been implicated in cardiovascular disorders, carcinogenesis and cancer progression. For functional and diagnostic studies on the HIF-1α protein, we have identified single-domain antibody fragments directed against this protein by using a llama-derived nonimmune phage display library. This library displays the variable domains of the heavy-chain antibody subclass, found in these animals. Phage display selection with six recombinant HIF-1α proteins yielded five different antibody fragments. By epitope-mapping, we show that all five antibody fragments bind within the functionally important oxygen-dependent degradation domain of the HIF-1α protein. Two of these antibody fragments were engineered into bivalent antibodies that were able to detect human HIF-1α by immunohistochemistry, Western blotting and immunoprecipitation, and mouse HIF-1α by immunofluorescence and immunoprecipitation. These are the first single-domain antibody fragments that may be used in exploration of HIF-1α as a possible therapeutic target through molecular applications.