Abstract:
Serpins (serine protease inhibitors) are a superfamily of over 500 proteins that are expressed in many organisms. Serpins inhibit serine proteases by a unique mechanism that results in the cleavage of the serpin and the formation of a stable covalent complex between the protease and the inhibitor. As a result both the protease and the serpin are inactivated. Neuroserpin is a serpin recently identified as an axonally transported and secreted protein from chicken dorsal root ganglions and subsequently shown to be expressed in the central and peripheral nervous systems. Analysis of the protein sequence of neuroserpin identified an arginine at the putative P1 position within the reactive centre loop sequence. This suggested that it was likely to inhibit trypsin-like serine proteases.
Our group identified neuroserpin expression in endocrine cells and found that it was likely to be targeted to the regulated secretory pathway (RSP). This result implicated neuroserpin as a regulator of enzymes involved in the maturation of prohormones. This thesis research investigated a role for neuroserpin as an inhibitor of trypsin-like serine proteases in the RSP. The research focussed on the prohormone convertase SPC3, a serine protease known to be sorted to the RSP in endocrine cells and capable of cleaving basic amino acids. Effects of neuroserpin on the biosynthesis of SPC3 as well as pro-opiomelanocortin (POMC) processing were investigated. Neuroserpin was shown to complex with the mature 64 kDa form of SPC3 but was unable to complex with the full length 86 kDa form. Complexation was seen under conditions of pH and calcium concentration consistent with those found later in the RSP, where the mature form of SPC3 is generated. The enzymatic activity of SPC3 was also inhibited by neuroserpin under similar conditions. However, the enzyme assays indicated a high molar ratio of neuroserpin to SPC3 was required, questioning the physiological role of this inhibition. These studies were complemented with cell culture studies in AtT-20 cells, a widely used model of prohormone processing. Neuroserpin and an engineered neuroserpin variant in which the P4 alanine was mutated to arginine (NSA359R) were stably expressed in AtT-20 cells. The variant was predicted to inhibit SPC3 and to possibly be a useful tool for analyses of prohormone and SPC3 biosynthesis. The effects of expression of these proteins on both SPC3 and POMC biosynthesis were determined by pulse-chase analysis and immunoprecipitation. No inhibition of the processing of either PC3 or POMC was detected. This suggested that neither neuroserpin nor NSA359R inhibited SPC3 nor any other enzyme that cleaved POMC to generate cleavage fragments recognized by the POMC site-specific antisera. However changes were seen in the levels of POMC expression. Published in vitro studies of protease inhibitory activity towards a number of commercially available proteases had suggested the most likely target protease of neuroserpin was tissue plasminogen activator (tPA). Therefore experiments were also undertaken on tPA which is known to sort to the RSP. Different molecular weight recombinant tPA:neuroserpin complexes were detected at pH 7.4 and 6.0, suggesting a role for neuroserpin as a regulator of tPA within the changing environment of the maturing secretory vesicle.
Other research in our group had found that altered expression of neuroserpin in AtT-20 cells and the phaeochromocytoma PC12 cell line resulted in altered neurite outgrowth. It was also observed that the adhesive behaviour of both cell lines appeared to have altered following changes in neuroserpin expression. Therefore, in the latter part of this thesis project the effects of neuroserpin on cell-matrix and cell-cell interactions were investigated. A number of cell lines were treated with exogenous recombinant neuroserpin for analysis of cell attachment behaviour. In light of the known interaction of the closely related serpin plasminogen activator inhibitor 1 (PAI-1) with the matrix protein vitronectin, an analogous interaction between neuroserpin and vitronectin was investigated. No interaction was detected. The expression of the α1 integrin subunit and the actin organisation in the PC12 cell lines with altered levels of neuroserpin were also examined. No differences were found. Scanning electron microscopic analysis of these cell lines revealed major morphological differences between the cell lines. Stable expression of a P1 variant neuroserpin (NSR362A), which is unable to complex with tPA, displayed a similar phenotype to cell lines over expressing wild type neuroserpin. Treatment of PC12 cell lines with exogenous recombinant neuroserpin resulted in a change in the phenotypes of under expressing and parent cell lines to resemble cells with higher expression levels. Analysis of the calcium dependence of cell-cell adhesion indicated that the cadherin family of adhesion molecules could be involved in neuroserpin's effects on cell adhesion. Studies of cadherin in the PC12 cell lines indicated neuronal-cadherin expression was correlated with neuroserpin or NSR362A expression in PC12 cells. These results suggest a non-inhibitory role for neuroserpin in the mediation of the adhesive interactions of nerve and endocrine cells and could indicate a role in synaptic stabilization, learning and memory.
Two models are proposed to guide further studies into neuroserpin's possible role(s) in cell-cell adhesion. Intracellular signaling pathways that may mediate these effects may also link to the observed effect of neuroserpin on POMC expression in AtT-20 cells.