D. Fritzinger - Academia.edu (original) (raw)

Papers by D. Fritzinger

Advances in Experimental Medicine and Biology, 2008

To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with l... more To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with less immunogenicity, we have prepared human C3/CVF hybrid proteins, in which the C-terminus of the !-chain of human C3 is exchanged with homologous regions of the C-terminus of the "-chain of CVF. We show that these hybrid 1496, is shown to be effective in reducing complement-mediated damage in two disease models in mice, collagen-induced arthritis and myocardial ischemia/reperfusion injury. 1 Background and Concept Cobra Venom Factor (CVF) has long been known to be a structural analog of a complement component C3 (Vogel 1991; Vogel et al. 1984, 1996). Both C3 and CVF are synthesized as single-chain pre-pro-proteins that are subsequently processed into the mature two-chain C3 protein, and the mature three-chain CVF protein, respectively (de Bruijn and Fey 1985; Fritzinger et al. 1994). The two proteins share extensive sequence similarity, at both the protein and DNA levels. CVF and human C3 are approximately 50% identical at the protein level and approximately 70% similar if one allows for conservative replacements (Fritzinger et al. 1994). The structural homology between cobra C3 and CVF is even greater;

Blood, 2009

Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the cli... more Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)–cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lym...

Advances in Experimental Medicine and Biology, 1996

Cobra Venom Factor (CVF) is an unusual venom component known to be present in the venom of the co... more Cobra Venom Factor (CVF) is an unusual venom component known to be present in the venom of the cobra species Naja, Ophiophagus, and Hemachatus of the Elapidae family (1). CVF is not a toxin in the classical sense. As a matter of fact, the purified molecule is not toxic. It specifically interacts with components of the serum complement system, leading to complement activation which in turn leads to the consumption of complement activity.

Infection and Immunity, 1999

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither w... more We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited ho...

Complement component C3 is a multifunctional protein known to interact specifically with more tha... more Complement component C3 is a multifunctional protein known to interact specifically with more than 10 different plasma proteins or cell surface receptors. Cobra venom contains cobra venom factor, a structural analogue of C3 that shares some properties with C3 (e.g., formation of a C3/C5 convertase) but differs in others (e.g., susceptibility to regulation by factors H and I). The elucidation of structural differences between C3 and cobra venom factor can be expected to help identify functionally important regions of C3 molecules. To that end we have undertaken the molecular cloning of both cobra C3 and cobra venom factor to take advantage of the unique biologic system where both proteins are produced by the same species. We report the primary structure of cobra C3 mRNA and the derived protein structure. Cobra C3 mRNA is 5211 bp in length. It contains an open reading frame of 4953 bp coding for a single pre-pro-C3 molecule, consisting of a 22-amino acid signal sequence, a 633-amino a...

The Open Pain Journal, 2016

Certain types of pain are major unmet medical needs that affect more than 8 percent of the popula... more Certain types of pain are major unmet medical needs that affect more than 8 percent of the population. Neuropathic pain can be caused by many pathogenic processes including injury, autoimmune disease, neurological disease, endocrine dysfunction, infection, toxin exposure, and substance abuse and is frequently resistant to available pain therapies. The same can be said of post-surgical pain, which can arise from uncontrolled inflammation around the wound site. The complement system is part of the innate immune system and can both initiate and sustain acute and chronic inflammatory pain. Here we review the complement system and original investigations that identify potential drug targets within this system. Drugs that act to inhibit the complement system could fill major gaps in our current standard of care for neuropathic pain states.

Hawaii Medical Journal, Jun 1, 2005

Molecular immunology, 2014

The complement system is an integral component of both innate and adaptive immunity. However, com... more The complement system is an integral component of both innate and adaptive immunity. However, complement is also a pathogenetic factor in many diseases. The development of agents for therapeutic complement inhibition is the topic of intense investigations by many investigators. We have developed a distinctly different therapeutic approach: complement depletion rather than inhibition. This approach is based on cobra venom factor (CVF), a C3 analog known to be able to safely deplete complement. This manuscript will briefly review the structure and activity of CVF, along with its similarities and differences to C3. Exploiting the knowledge of the structure/function relationship of CVF and C3, we created derivatives of human C3 which display the CVF-like activity of depleting complement, referred to as humanized CVF (hCVF). This review describes the structure and activity of hCVF, including the important property of not cleaving C5. The efficacy of hCVF for therapeutic complement deplet...

Hawaii medical journal, 2005

Molecular Immunology, 2010

interaction there are extended exosite interactions between the two MASP-2 molecules, as well. Ex... more interaction there are extended exosite interactions between the two MASP-2 molecules, as well. Exploring these exosite regions could help us in revealing the mechanism of the autoactivation as well as in understanding the high selectivity of MASP-2 and related early serine protease enzymes (C1r, C1s, MASP-1) of the classical and the lectin pathways.

Toxicon, 2012

Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally... more Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally and functionally highly homologous to complement component C3. CVF, like C3b, the activated form of C3, forms a bimolecular complex with Factor B in serum, called C3/C5 convertase, an enzyme which activates complement components C3 and C5. Despite the high degree of homology, the two C3/C5 convertases exhibit significant functional differences. The most important difference is that the convertase formed with CVF (CVF,Bb) is physico-chemically far more stable than the convertase formed with C3b (C3b,Bb). In addition, the CVF,Bb convertase and CVF are completely resistant to inactivation by the complement regulatory proteins Factor H and Factor I. Furthermore, the CVF,Bb enzyme shows efficient C5-cleaving activity in fluid phase. In contrast, the C3b,Bb enzyme is essentially devoid of fluid-phase C5-cleaving activity. By taking advantage of the high degree of sequence identity at both the amino acid (85%) and DNA levels (93%) between CVF and cobra C3, we created hybrid proteins of CVF and cobra C3 where sections, or only a few amino acids, of the CVF sequence were replaced with the homologous amino acid sequence of cobra C3. In a first set of experiments, we created five hybrid proteins, termed H1 through H5, where the cobra C3 substitutions collectively spanned the entire length of the CVF protein. We also created three additional hybrid proteins where only four or five amino acid residues in CVF were exchanged with the corresponding amino acid residues from cobra C3. Collectively, these hybrid proteins, representing loss-of-function mutants of CVF, allowed the identification of regions and individual amino acid residues important for the CVF-specific functions. The results include the observation that the CVF β-chain is crucially important for forming a stable convertase, whereas the CVF α-chain appears to harbor no CVF-specific functions. Furthermore, the CVF γ-chain is additionally important for the fluid-phase C5-cleaving activity of CVF,Bb. Interestingly, the structural changes in the individual hybrid proteins differentially affected the molecular functions of the CVF,Bb enzyme such as convertase formation, C3 cleavage, and C5 cleavage.

Molecular Immunology, 2010

Advances in Experimental Medicine and Biology, 2008

To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with l... more To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with less immunogenicity, we have prepared human C3/CVF hybrid proteins, in which the C-terminus of the !-chain of human C3 is exchanged with homologous regions of the C-terminus of the "-chain of CVF. We show that these hybrid 1496, is shown to be effective in reducing complement-mediated damage in two disease models in mice, collagen-induced arthritis and myocardial ischemia/reperfusion injury. 1 Background and Concept Cobra Venom Factor (CVF) has long been known to be a structural analog of a complement component C3 (Vogel 1991; Vogel et al. 1984, 1996). Both C3 and CVF are synthesized as single-chain pre-pro-proteins that are subsequently processed into the mature two-chain C3 protein, and the mature three-chain CVF protein, respectively (de Bruijn and Fey 1985; Fritzinger et al. 1994). The two proteins share extensive sequence similarity, at both the protein and DNA levels. CVF and human C3 are approximately 50% identical at the protein level and approximately 70% similar if one allows for conservative replacements (Fritzinger et al. 1994). The structural homology between cobra C3 and CVF is even greater;

Blood, 2009

Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the cli... more Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)–cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lym...

Advances in Experimental Medicine and Biology, 1996

Cobra Venom Factor (CVF) is an unusual venom component known to be present in the venom of the co... more Cobra Venom Factor (CVF) is an unusual venom component known to be present in the venom of the cobra species Naja, Ophiophagus, and Hemachatus of the Elapidae family (1). CVF is not a toxin in the classical sense. As a matter of fact, the purified molecule is not toxic. It specifically interacts with components of the serum complement system, leading to complement activation which in turn leads to the consumption of complement activity.

Infection and Immunity, 1999

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither w... more We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited ho...

Complement component C3 is a multifunctional protein known to interact specifically with more tha... more Complement component C3 is a multifunctional protein known to interact specifically with more than 10 different plasma proteins or cell surface receptors. Cobra venom contains cobra venom factor, a structural analogue of C3 that shares some properties with C3 (e.g., formation of a C3/C5 convertase) but differs in others (e.g., susceptibility to regulation by factors H and I). The elucidation of structural differences between C3 and cobra venom factor can be expected to help identify functionally important regions of C3 molecules. To that end we have undertaken the molecular cloning of both cobra C3 and cobra venom factor to take advantage of the unique biologic system where both proteins are produced by the same species. We report the primary structure of cobra C3 mRNA and the derived protein structure. Cobra C3 mRNA is 5211 bp in length. It contains an open reading frame of 4953 bp coding for a single pre-pro-C3 molecule, consisting of a 22-amino acid signal sequence, a 633-amino a...

The Open Pain Journal, 2016

Certain types of pain are major unmet medical needs that affect more than 8 percent of the popula... more Certain types of pain are major unmet medical needs that affect more than 8 percent of the population. Neuropathic pain can be caused by many pathogenic processes including injury, autoimmune disease, neurological disease, endocrine dysfunction, infection, toxin exposure, and substance abuse and is frequently resistant to available pain therapies. The same can be said of post-surgical pain, which can arise from uncontrolled inflammation around the wound site. The complement system is part of the innate immune system and can both initiate and sustain acute and chronic inflammatory pain. Here we review the complement system and original investigations that identify potential drug targets within this system. Drugs that act to inhibit the complement system could fill major gaps in our current standard of care for neuropathic pain states.

Hawaii Medical Journal, Jun 1, 2005

Molecular immunology, 2014

The complement system is an integral component of both innate and adaptive immunity. However, com... more The complement system is an integral component of both innate and adaptive immunity. However, complement is also a pathogenetic factor in many diseases. The development of agents for therapeutic complement inhibition is the topic of intense investigations by many investigators. We have developed a distinctly different therapeutic approach: complement depletion rather than inhibition. This approach is based on cobra venom factor (CVF), a C3 analog known to be able to safely deplete complement. This manuscript will briefly review the structure and activity of CVF, along with its similarities and differences to C3. Exploiting the knowledge of the structure/function relationship of CVF and C3, we created derivatives of human C3 which display the CVF-like activity of depleting complement, referred to as humanized CVF (hCVF). This review describes the structure and activity of hCVF, including the important property of not cleaving C5. The efficacy of hCVF for therapeutic complement deplet...

Hawaii medical journal, 2005

Molecular Immunology, 2010

interaction there are extended exosite interactions between the two MASP-2 molecules, as well. Ex... more interaction there are extended exosite interactions between the two MASP-2 molecules, as well. Exploring these exosite regions could help us in revealing the mechanism of the autoactivation as well as in understanding the high selectivity of MASP-2 and related early serine protease enzymes (C1r, C1s, MASP-1) of the classical and the lectin pathways.

Toxicon, 2012

Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally... more Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally and functionally highly homologous to complement component C3. CVF, like C3b, the activated form of C3, forms a bimolecular complex with Factor B in serum, called C3/C5 convertase, an enzyme which activates complement components C3 and C5. Despite the high degree of homology, the two C3/C5 convertases exhibit significant functional differences. The most important difference is that the convertase formed with CVF (CVF,Bb) is physico-chemically far more stable than the convertase formed with C3b (C3b,Bb). In addition, the CVF,Bb convertase and CVF are completely resistant to inactivation by the complement regulatory proteins Factor H and Factor I. Furthermore, the CVF,Bb enzyme shows efficient C5-cleaving activity in fluid phase. In contrast, the C3b,Bb enzyme is essentially devoid of fluid-phase C5-cleaving activity. By taking advantage of the high degree of sequence identity at both the amino acid (85%) and DNA levels (93%) between CVF and cobra C3, we created hybrid proteins of CVF and cobra C3 where sections, or only a few amino acids, of the CVF sequence were replaced with the homologous amino acid sequence of cobra C3. In a first set of experiments, we created five hybrid proteins, termed H1 through H5, where the cobra C3 substitutions collectively spanned the entire length of the CVF protein. We also created three additional hybrid proteins where only four or five amino acid residues in CVF were exchanged with the corresponding amino acid residues from cobra C3. Collectively, these hybrid proteins, representing loss-of-function mutants of CVF, allowed the identification of regions and individual amino acid residues important for the CVF-specific functions. The results include the observation that the CVF β-chain is crucially important for forming a stable convertase, whereas the CVF α-chain appears to harbor no CVF-specific functions. Furthermore, the CVF γ-chain is additionally important for the fluid-phase C5-cleaving activity of CVF,Bb. Interestingly, the structural changes in the individual hybrid proteins differentially affected the molecular functions of the CVF,Bb enzyme such as convertase formation, C3 cleavage, and C5 cleavage.

Molecular Immunology, 2010