Normalt sett skyddar komplementsystemet kroppen mot infektioner och patogener. Vid vissa typer av njursjukdomar, framför allt vid C3-glomerulopati, förekommer autoantikroppar som kallas ”nephritic factors” (NeF). Sådana antikroppar stabiliserar enzymkomplex (konvertas) i komplementsystemet, vilket leder till destruktiv komplementaktivering via den alternativa vägen. Syftet med studien var att utveckla minst en metod för att analysera C5NeF på kliniska prover.  C5NeF In-House ELISA analyserade bindning av C5NeF till C5-konvertas. Analys av C5-klyvning i löslig fas kvantifierade mängden C5a som bildats vid stabilisering av C5-konvertas. Cut-off för analyserna bestämdes genom analys av prover från 20 friska blodgivare. Tolv patientprover med möjlig förekomst av C5NeF analyserades. För att utesluta falskt positiv reaktion i C5NeF in-house ELISA analyserades även förekomst av antikroppar mot specifika enskilda komplementproteiner. Åtta patientprover var positiva i C5NeF In-House ELISA, fem patientprover uppvisade positivt resultat för C3NeF, vilket inte var oväntat utifrån tidigare publikationer som visat att det är vanligt att patienter med C5NeF också ofta är positiva för C3NeF. Tre patientprover erhöll positivt resultat i endast C5NeF In-House ELISA och två av dessa var positiva i analys av C5-klyvning i löslig fas. Studien resulterade i etablering av en metod för analys av C5NeF. / Normally the complement system protects the body from infections and pathogens. In certain types of kidney diseases, mainly C3-glomerulopathy, autoantibodies called ”Nephritic Factors” (NeF) are found. NeFs stabilize enzyme complexes (convertases) in the complement system, an event which leads to destructive complement activation via the alternative pathway. The purpose of this study was to develop at least one method to analyse C5NeF on clinical samples.  C5NeF In-House ELISA analysed binding of C5NeF to C5 convertases. Analysis of C5-cleavage in the soluble phase measured the amount of C5a formed when C5-convertase was stabilized. Cut-off for the analyses was determined through analysis of 20 blood donor samples from healthy individuals. Twelve patient samples with possible C5NeF were analysed. To exclude false positive results in C5NeF In-House ELISA analysis of antibodies against specific single complement factors was performed.  Eight patient samples were positive in C5NeF In-House ELISA, five patient samples showed positive result for C3NeF, a finding which was not unexpected as previous publications have shown that concomitant presence of C3NeF and C5NeF is common in C3-glomerulopathy. Where most patients are positive for both C3NeF and C5NeF. Three patient samples received positive result in only C5NeF In-House ELISA and two of these samples were positive in the analysis of C5-cleavage in soluble phase. In conclusion, in this study a method to examine C5NeF was developed.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) is the ninth member of the Ca+2-dependent mammalian proprotein convertase super family of serine endoproteases that is structurally related to the bacterial subtilisin and yeast kexin enzymes. It plays a critical role in the regulation of lipid metabolism and cholesterol homeostasis by binding to and degrading low-density lipoprotein-receptor (LDL-R) which is responsible for the clearance of circulatory LDL-cholesterol from the blood. Owing to this functional property, there is plenty of research interest in the development of functional inhibitors of PCSK9 which may find important biochemical applications as therapeutic agents for lowering plasma LDL-cholesterol. The catalytic domain of PCSK9 binds to the EGF-A domain of LDL-R on the cell surface to form a stable complex and re-routes the receptor from its normal endosomal recycling pathway to the lysosomal compartments leading to its degradation. Owing to these findings, we propose that selected peptides from PCSK9 catalytic domain, particularly its disulphide (S-S) bridged loop1 323-358 and loop2 365-385, are likely to exhibit strong affinity towards the EGF-A domain of LDL-R. Several regular peptides along with corresponding all- dextro and retro-inverse peptides as well as the gain-of-function mutant variants were designed and tested for their regulatory effects towards LDL-R expression and PCSK9-binding in human hepatic HepG2 and mouse hepatic Hepa1c1c7 cells. Our data indicated that disulfide bridged loop1-hPCSK9323-358 and its H357 mutant as well as two short loop2-hPCSK9372-380 and its Y374 mutant peptides modestly promote the LDL-R protein levels. Our study concludes that specific peptides from the PCSK9 catalytic domain can regulate LDL-R and may be useful for development of novel class of therapeutic agents for cholesterol regulation.