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Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics.
Schaer, Christian A; Owczarek, Catherine; Deuel, Jeremy W; Schauer, Stefan; Baek, Jin Hyen; Yalamanoglu, Ayla; Hardy, Matthew P; Scotney, Pierre D; Schmidt, Peter M; Pelzing, Matthias; Soupourmas, Peter; Buehler, Paul W; Schaer, Dominik J.
Afiliação
  • Schaer CA; Division of Internal Medicine, University Hospital Zurich, Ramistrasse 100, CH-8091, Zurich, Switzerland.
  • Owczarek C; CSL Limited, Bio21 Institute, Parkville, Australia.
  • Deuel JW; Division of Internal Medicine, University Hospital Zurich, Ramistrasse 100, CH-8091, Zurich, Switzerland.
  • Schauer S; Functional Genomics Center Zurich, University of Zurich, Zurich, Switzerland.
  • Baek JH; Division of Blood Components and Devices, Office of Blood Research and Review, Laboratory of Biochemistry and Vascular Biology, FDA, Sliver Spring, MD, USA.
  • Yalamanoglu A; Division of Blood Components and Devices, Office of Blood Research and Review, Laboratory of Biochemistry and Vascular Biology, FDA, Sliver Spring, MD, USA.
  • Hardy MP; CSL Limited, Bio21 Institute, Parkville, Australia.
  • Scotney PD; CSL Limited, Bio21 Institute, Parkville, Australia.
  • Schmidt PM; CSL Limited, Bio21 Institute, Parkville, Australia.
  • Pelzing M; CSL Limited, Bio21 Institute, Parkville, Australia.
  • Soupourmas P; CSL Limited, Bio21 Institute, Parkville, Australia.
  • Buehler PW; Division of Blood Components and Devices, Office of Blood Research and Review, Laboratory of Biochemistry and Vascular Biology, FDA, Sliver Spring, MD, USA.
  • Schaer DJ; Division of Internal Medicine, University Hospital Zurich, Ramistrasse 100, CH-8091, Zurich, Switzerland. Dominik.Schaer@usz.ch.
BMC Biotechnol ; 18(1): 15, 2018 03 15.
Article em En | MEDLINE | ID: mdl-29544494
BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Haptoglobinas / Hemoglobinas / Serina Endopeptidases / Engenharia de Proteínas Limite: Animals / Humans / Male Idioma: En Ano de publicação: 2018 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Haptoglobinas / Hemoglobinas / Serina Endopeptidases / Engenharia de Proteínas Limite: Animals / Humans / Male Idioma: En Ano de publicação: 2018 Tipo de documento: Article