An emerging number of diseases and therapeutic approaches with defined involvement of the complem... more An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.
Biochemical and biophysical research communications, Jan 19, 2015
CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by ... more CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by acting on the complement C3 convertases. CD55 is widely localized in the cholesterol rich regions of the cell plasma membrane termed membrane rafts. CD55 is attached to these specialized regions via a GPI link on the outer leaflet of the plasma membrane. Membrane rafts anchor many important signaling proteins, which control several cellular functions within the cell. For example, we recently demonstrated that the membrane raft protein and complement inhibitor CD59 also controls insulin secretion by an intracellular mechanism. Therefore, we have in this study aimed at addressing the expression and function of CD55 in pancreatic beta cells. To this end, we observe that CD55 is highly expressed in INS1 832/13 beta cells as well as human pancreatic islets. Diabetic human islets show a tendency for increased expression of CD55 when compared to the healthy controls. Importantly, silencing of C...
Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune ... more Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harbour...
Advances in experimental medicine and biology, 2007
The complement system is an important part of the innate immune system providing immediate protec... more The complement system is an important part of the innate immune system providing immediate protection against pathogens without requirement for previous exposure. Complement also serves an important role in priming the adaptive immune response through opsonization, ...
Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which c... more Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which carries an 8 kDa chondroitin sulphate chain, and heavy chain 3 (H3) of 74 kDa. The two polypeptides are linked through an ester bond between an internal N-acetylgalactosamine residue of the chondroitin sulphate chain and the C-terminal aspartic acid residue of H3. Both bikunin and H3 are synthesized by hepatocytes and become linked as they pass through the Golgi complex. H3 is synthesized with both N- and C-terminal extensions which are released during intracellular transport. To be able to analyse the assembly of pre-alpha-inhibitor in detail, we have cloned and sequenced the cDNA of rat H3. Upon expression of the protein in COS-1 cells, both propeptides were found to be released. Furthermore, co-expression of H3 and bikunin resulted in the two polypeptides becoming coupled, indicating that cells other than hepatocytes may have the capacity to form chondroitin sulphate-containing links.
Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classifie... more Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecti...
Apoptotic cells have been reported to down-regulate membrane-bound complement regulatory proteins... more Apoptotic cells have been reported to down-regulate membrane-bound complement regulatory proteins (m-C-Reg) and to activate complement. Nonetheless, most apoptotic cells do not undergo complement-mediated lysis. Therefore, we hypothesized that fluid phase complement inhibitors would bind to apoptotic cells and compensate functionally for the loss of m-C-Reg. We observed that m-C-Reg are down-regulated rapidly upon apoptosis but that complement activation follows only after a gap of several hours. Coinciding with, but independent from, complement activation, fluid phase complement inhibitors C4b-binding protein (C4BP) and factor H (fH) bind to the cells. C4BP and fH do not entirely prevent complement activation but strongly limit C3 and C9 deposition. Late apoptotic cells, present in blood of healthy controls and systemic lupus erythematosus patients, are also positive for C4BP and fH. Upon culture, the percentage of late apoptotic cells increases, paralleled by increased C4BP binding. C4BP binds to dead cells mainly via phosphatidylserine, whereas fH binds via multiple interactions with CRP playing no major role for binding of C4BP or fH. In conclusion, during late apoptosis, cells acquire fluid phase complement inhibitors that compensate for the down-regulation of m-C-Reg and protect against excessive complement activation and lysis.
An emerging number of diseases and therapeutic approaches with defined involvement of the complem... more An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.
Biochemical and biophysical research communications, Jan 19, 2015
CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by ... more CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by acting on the complement C3 convertases. CD55 is widely localized in the cholesterol rich regions of the cell plasma membrane termed membrane rafts. CD55 is attached to these specialized regions via a GPI link on the outer leaflet of the plasma membrane. Membrane rafts anchor many important signaling proteins, which control several cellular functions within the cell. For example, we recently demonstrated that the membrane raft protein and complement inhibitor CD59 also controls insulin secretion by an intracellular mechanism. Therefore, we have in this study aimed at addressing the expression and function of CD55 in pancreatic beta cells. To this end, we observe that CD55 is highly expressed in INS1 832/13 beta cells as well as human pancreatic islets. Diabetic human islets show a tendency for increased expression of CD55 when compared to the healthy controls. Importantly, silencing of C...
Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune ... more Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harbour...
Advances in experimental medicine and biology, 2007
The complement system is an important part of the innate immune system providing immediate protec... more The complement system is an important part of the innate immune system providing immediate protection against pathogens without requirement for previous exposure. Complement also serves an important role in priming the adaptive immune response through opsonization, ...
Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which c... more Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which carries an 8 kDa chondroitin sulphate chain, and heavy chain 3 (H3) of 74 kDa. The two polypeptides are linked through an ester bond between an internal N-acetylgalactosamine residue of the chondroitin sulphate chain and the C-terminal aspartic acid residue of H3. Both bikunin and H3 are synthesized by hepatocytes and become linked as they pass through the Golgi complex. H3 is synthesized with both N- and C-terminal extensions which are released during intracellular transport. To be able to analyse the assembly of pre-alpha-inhibitor in detail, we have cloned and sequenced the cDNA of rat H3. Upon expression of the protein in COS-1 cells, both propeptides were found to be released. Furthermore, co-expression of H3 and bikunin resulted in the two polypeptides becoming coupled, indicating that cells other than hepatocytes may have the capacity to form chondroitin sulphate-containing links.
Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classifie... more Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecti...
Apoptotic cells have been reported to down-regulate membrane-bound complement regulatory proteins... more Apoptotic cells have been reported to down-regulate membrane-bound complement regulatory proteins (m-C-Reg) and to activate complement. Nonetheless, most apoptotic cells do not undergo complement-mediated lysis. Therefore, we hypothesized that fluid phase complement inhibitors would bind to apoptotic cells and compensate functionally for the loss of m-C-Reg. We observed that m-C-Reg are down-regulated rapidly upon apoptosis but that complement activation follows only after a gap of several hours. Coinciding with, but independent from, complement activation, fluid phase complement inhibitors C4b-binding protein (C4BP) and factor H (fH) bind to the cells. C4BP and fH do not entirely prevent complement activation but strongly limit C3 and C9 deposition. Late apoptotic cells, present in blood of healthy controls and systemic lupus erythematosus patients, are also positive for C4BP and fH. Upon culture, the percentage of late apoptotic cells increases, paralleled by increased C4BP binding. C4BP binds to dead cells mainly via phosphatidylserine, whereas fH binds via multiple interactions with CRP playing no major role for binding of C4BP or fH. In conclusion, during late apoptosis, cells acquire fluid phase complement inhibitors that compensate for the down-regulation of m-C-Reg and protect against excessive complement activation and lysis.
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Papers by Anna Blom