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1.
Biochemistry ; 48(49): 11755-65, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19842711

RESUMO

Betaglycan is a coreceptor for members of the transforming growth factor beta (TGF-beta) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Delta10 and Sol Delta11, bind TGF-beta, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-beta activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has K(d)'s in the low nanomolar range for the three TGF-beta isoforms, while those for Sol Delta10 and Sol Delta11 are 1-2 orders of magnitude higher. SPR analysis further shows that the K(d)'s of Sol Delta11 are not changed in the presence of Sol Delta10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-beta through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan's TGF-beta neutralizing activity.


Assuntos
Testes de Neutralização , Fragmentos de Peptídeos/metabolismo , Proteoglicanas/química , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Fibrinolisina/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteoglicanas/antagonistas & inibidores , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/química
2.
Am J Physiol Renal Physiol ; 292(1): F321-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16954341

RESUMO

Transforming growth factor-beta (TGF-beta) is a key mediator in the pathogenesis of renal diseases. Betaglycan, also known as the type III TGF-beta receptor, regulates TGF-beta action by modulating its access to the type I and II receptors. Betaglycan potentiates TGF-beta; however, soluble betaglycan, which is produced by the shedding of the membrane-bound receptor, is a potent antagonist of TGF-beta. In the present work, we have used a recombinant form of soluble betaglycan (SBG) to prevent renal damage in genetically obese and diabetic db/db mice. Eight-wk-old db/db or nondiabetic (db/m) mice were injected intraperitoneally with 50 mug of SBG or vehicle alone three times a wk for 8 wk. The db/db mice that received vehicle presented albuminuria and increased serum creatinine, as well as glomerular mesangial matrix expansion. The db/db mice treated with SBG exhibited a reduction in serum creatinine, albuminuria, and structural renal damage. These effects were associated with lower kidney levels of mRNAs encoding TGF-beta1, TGF-beta2, TGF-beta3, collagen IV, collagen I, fibronectin, and serum glucocorticoid kinase as well as a reduction in the immunostaining of collagen IV and fibronectin. Our data indicate that SBG is a renoprotective agent that neutralized TGF-beta actions in this model of nephropathy. Because SBG has a high affinity for all TGF-beta isoforms, in particular TGF-beta2, it is found naturally in serum and tissues and its shedding may be regulated. We believe that SBG shall prove convenient for long-term treatment of kidney diseases and other pathologies in which TGF-beta plays a pathophysiological role.


Assuntos
Nefropatias/induzido quimicamente , Nefropatias/patologia , Polissacarídeos/uso terapêutico , Albuminúria/urina , Animais , Colágeno/biossíntese , Creatinina/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Fibronectinas/metabolismo , Mesângio Glomerular/patologia , Imuno-Histoquímica , Nefropatias/genética , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Fator de Crescimento Transformador beta/antagonistas & inibidores
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