Daniel J. Knauer

Epidermal growth factor receptor immunoreactivity in rat brain. Development and cellular localization

In rat brain, distinct epidermal growth factor-receptor immunoreactivity (EGFR-IR) first appeared in astroglia at about day 16 postnatal, reached maximum intensity at 19 days and then became much weaker as the animals reached adulthood. EGFR-IR was also observed in cerebellar Purkinje cells as early as 11 days postnatal and was maintained into adulthood. In adult and aged animals the most prominent EGF receptor immunostaining occurred in cerebral cortex neurons (layers IV and V) that had the morphology of basket cells. Immunoreactive neurons were abundant in the cingulate, frontal, frontoparietal and striate cortices. In the frontoparietal cortex, EGFR positive neurons were most numerous in the motor area, diminishing laterally towards the somatosensory area. The localization and time of appearance of EGFR-IR did not appear consistent with a direct mitogenic role of the EGF domain in astroglia proliferation during development. However, the EGFR may be involved in neuronal survival and/or neuron-glia signalling.

Epidermal growth factor receptor immunoreactivity in rat brain astrocytes. Response to injury

Brain astroglia in normal adult rats stained weakly or not at all with an antibody to epidermal growth factor receptor (EGFR). A dramatic change took place after injury. The astrocytes adjacent to an entorhinal ablation and in deafferented areas of the hippocampus showed prominent EGFR immunoreactivity. Cells that were EFGR-immunoreactive also stained intensely with an antibody to glial fibrillary acidic protein (GFAP). The localization and the time course of appearance of EGFR/GFAP immunoreactivity suggests that EGFR may be involved in the conversion of a normal into a reactive astrocyte.

Identification of a protease inhibitor produced by astrocytes that is structurally and functionally homologous to human protease nexin-I

In the present studies we have compared the structural and biochemical properties of human protease nexin-I (PN-I) and a protease inhibitor present in the serum-free culture fluid of normal rat brain astrocytes. The inhibitor binds to and forms covalent complexes with human urokinase and thrombin. The inhibitor has an approximate Mr = 43,000 based on the size of the complexes (deduced from SDS-PAGE) and mediates the cellular binding and uptake of the proteases to which it links. Binding is heparin sensitive and occurs on a cell surface receptor that also binds complexes formed between proteases and a well-characterized cell-secreted protease inhibitor, human PN-I. In addition, the inhibitor co-migrates with PN-I on SDS-PAGE and cross-reacts with anti-PN-I antibody on immunoblots. A similar molecule, designated NPF, is produced by C6 glioma cells in culture and has neurite promoting activity on a neuroblastoma cell line.

The efficient catabolism of thrombin-protease nexin 1 complexes is a synergistic mechanism that requires both the LDL receptor-related protein and cell surface heparins.

Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5- to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis.

Protease nexins: cell-secreted proteins which regulate extracellular serine proteases

Abstract Cultured normal human fibroblasts release three different proteins called protease nexins into their medium which selectively form covalent linkages with certain serine proteases. These protease-protease nexin complexes then bind to cells, are internalized and degraded. For the protease thrombin, it has been shown that this process modulates its mitogenic action on the cells.

Requirement of lysine residues outside of the proposed pentasaccharide binding region for high affinity heparin binding and activation of human antithrombin III.

Variant forms of human antithrombin III with glutamine or threonine substitutions at Lys114, Lys125, Lys133, Lys136, and Lys139 were expressed in insect cells to evaluate their roles in heparin binding and activation. Recombinant native ATIII and all of the variants had very similar second order rate constants for thrombin inhibition in the absence of heparin, ranging from 1.13 × 105 M−1min−1 to 1.66 × 105 M−1min−1. Direct binding studies using 125I-flouresceinamine-heparin yielded a Kd of 6 nM for the recombinant native ATIII and K136T, whereas K114Q and K139Q bound heparin so poorly that a Kd could not be determined. K125Q had a moderately reduced affinity. Heparin binding affinity correlated directly with heparin cofactor activity. Recombinant native ATIII was nearly identical to plasma-purified ATIII, whereas K114Q and K139Q were severely impaired in heparin cofactor activity. K125Q and K136T were only slightly impaired. Based on these data, Lys114 and Lys139, which are outside of the putative pentasaccharide binding site, play pivotal roles in the high affinity binding of heparin to ATIII and the activation of thrombin inhibitory activity.

Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein

We recently identified a synthetic peptide, Pro47–Ile58, derived from the mature protease nexin 1 (PN1) sequence, that inhibited the low density lipoprotein receptor-related protein (LRP)-mediated internalization of thrombin-PN1 (Th-PN1) complexes. Presently, we have analyzed this sequence in Th-PN1 complex catabolism using two independent approaches: 1) An antibody was generated against Pro47–Ile58, which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface heparins. This places the structural determinant in PN1 mediating complex internalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His48, or two Ala substitutions, one at His48 and another at Asp49, were expressed in Sf9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15%, respectively, when compared with the catabolic rate of native Th-PN1 complexes. This is the first analysis of a structural determinant in a serineprotease inhibitor (SERPIN) required for LRP-mediated internalization and in part may explain the cryptic nature of this site in the unreacted serine protease inhibitor.

Protease Nexins: Secreted Protease Inhibitors That Regulate Protease Activity at or near the Cell Surface

Publisher Summary This chapter provides an overview of the secreted protease inhibitors that regulate protease activity at or near the cell surface. Proteolytic enzymes are proteins that could carry out digestive functions and metabolize the proteins of the organisms in which they reside. These digestive proteases have acquired a higher degree of specialization by restricting their action to a limited number of peptide bonds. As a result of this specialization, certain proteases arose with high specificities that could cleave only certain proteins. This limited proteolysis does not destroy the protein substrate, but results in fragments with altered properties, providing a basis for the regulation of important physiological processes. Specific regulatory proteases play key roles in the control of blood coagulation and clot formation, platelet aggregation and release, fibrinolysis, complement activation, hormone processing, transport of secretory proteins across membranes, mammalian reproductive processes, viral assembly, and initiation of cell division. The PNs are protease inhibitors that could regulate protease activity at the surface of cells and in their immediate vicinity. By inhibiting the action of certain regulatory proteases at the cellular level, the PNs could limit the action of these proteases to specific sites for controlled times. This could provide an important modulation of cellular activities that are controlled by proteolytic enzymes.

Mechanism of Thrombin Clearance by Human Astrocytoma Cells

Abstract : Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimers disease, resulting in an accumulation of thrombin : protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin : PN1 complexes efficiently by a PN1‐dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor‐associated protein, an antagonist of the low‐density lipoprotein receptor‐related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell‐surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner : thrombin is first complexed with PN1, then bound to cell‐surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimers disease such as LRP.

Ligand blotting with 125I-fluoresceinamine-heparin

A highly sensitive method for ligand blotting with heparin has been developed. This ligand-blotting method is successful largely due to the ability to prepare heparin derivatives of high radiospecific activity. Heparin was modified with fluoresceinamine according to the method of C.G. Glabe, P.K. Harty, and S.D. Rosen [1983) Anal. Biochem. 130, 287-294), and this fluoresceinamine-derivatized heparin can be radioiodinated to a specific activity of 100,000 cmp/ng of uronic acid. This is a 500-fold increase in specific activity over Bolton-Hunter-modified heparin, as prepared by A.D. Cardin, K.R. Witt, and R.L. Jackson [1984) Anal. Biochem. 137, 368-373). 125I-Fluoresceinamine-derivatized heparin retains its ability to interact specifically with heparin-binding proteins such as human protease nexin-I and antithrombin III. 125I-Fluoresceinamine-derivatized heparin can be used to visualize and quantify heparin binding proteins on nitrocellulose. Protease nexin-I can be visualized at the nanogram level. In addition, ligand blotting with 125I-fluoresceinamine heparin can be combined with Cleveland digestion (D.W. Cleveland, S. Fisher, M.W. Kirschner, and U.K. Laemmli (1977) J. Biol. Chem. 252, 1102-1106) in order to identify heparin binding fragments of proteins with heparin binding domains.