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1.
Angew Chem Int Ed Engl ; 60(49): 25922-25932, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34523784

RESUMO

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.


Assuntos
Eritropoetina/metabolismo , Ácido N-Acetilneuramínico/biossíntese , Ácido N-Acetilneuramínico/síntese química , Sialiltransferases/metabolismo , Eritropoetina/química , Glicosilação , Humanos , Estrutura Molecular , Ácido N-Acetilneuramínico/química , Photobacterium/enzimologia , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
2.
J Pept Sci ; 27(1): e3283, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32885544

RESUMO

A convergent synthesis for erythropoietin (EPO) 1-28 N-glycopeptide hydrazides was developed. In this approach, EPO 1-28 peptides were synthesized on the solid phase and converted to C-terminal hydrazides after cleavage from the resin. After selective deprotection of the Asp24 side chain, the desired glycosylamine was coupled by pseudoproline-assisted Lansbury aspartylation. Although the initial yields of the EPO 1-28 glycopeptides were satisfactory, they could be markedly improved by increasing the purity of the peptide using a reversed-phase high-performance liquid chromatography (RP-HPLC) purification of the protected peptide.


Assuntos
Eritropoetina/química , Glicopeptídeos/química , Cromatografia Líquida de Alta Pressão , Técnicas de Síntese em Fase Sólida
3.
Angew Chem Int Ed Engl ; 56(19): 5252-5257, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28378443

RESUMO

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid-phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid-binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N-glycan.


Assuntos
Carboidratos/química , Saposinas/síntese química , Saposinas/metabolismo , Configuração de Carboidratos , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Saposinas/química
4.
PLoS Biol ; 15(1): e2000080, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28060820

RESUMO

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.


Assuntos
Proteólise , Receptores de Interleucina-6/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Glicosilação , Humanos , Espaço Intracelular/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Mutação/genética , Polissacarídeos/metabolismo , Prolina/metabolismo , Domínios Proteicos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Transdução de Sinais , Solubilidade , Valina/metabolismo
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