RESUMO
The asialoglycoprotein receptor (ASGP-R) is an abundant, carbohydrate-specific, endocytic receptor expressed by parenchymal cells of the liver. We recently demonstrated that the ASGP-R mediates the clearance of glycoproteins bearing Siaalpha2,6GalNAc as well as those bearing terminal Gal or GalNAc. We now report that glycoproteins such as haptoglobin, serum amyloid protein (SAP), and carboxylesterase that bear oligosaccharides with terminal Siaalpha2,6Gal are elevated in the plasma of ASGP-R-deficient mice. Because of their abundance in plasma, glycoproteins bearing terminal Siaalpha2,6Gal will saturate the ASGP-R and compete with each other on the basis of their relative affinities for the ASGP-R and their relative abundance. We propose that the ASGP-R mediates the clearance of glycoproteins that bear oligosaccharides terminating with Siaalpha2,6Gal and thereby helps maintain the relative concentrations of these glycoproteins in the blood.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Proteínas Sanguíneas/metabolismo , Glicoproteínas/sangue , Fígado/metabolismo , Oligossacarídeos/sangue , Ácidos Siálicos/sangue , Animais , Receptor de Asialoglicoproteína/genética , Proteínas Sanguíneas/genética , Glicoproteínas/genética , Fígado/citologia , Camundongos , Camundongos Knockout , Oligossacarídeos/genética , Ácidos Siálicos/genéticaRESUMO
Endogenous ligands have not, to date, been identified for the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed by parenchymal cells in the liver of mammals. On the basis of the rapid clearance of BSA bearing multiple chemically coupled sialic acid (Sia)alpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man tetrasaccharides (SiaGGnM-BSA) from the circulation, and the ability of the ASGP-R hepatic lectin-1 subunit to bind SiaGGnM-BSA, we previously proposed that glycoproteins modified with structures terminating with Siaalpha2,6GalNAc may represent previously unrecognized examples of endogenous ligands for this receptor. Here, we have taken a genetic approach using wild-type and ASGP-R-deficient mice to determine that the ASGP-R in vivo does indeed account for the rapid clearance of glycoconjugates terminating with Siaalpha2,6GalNAc. We have also determined that the ASGP-R is able to bind core-substituted oligosaccharides with the terminal sequence Siaalpha2,6Galbeta1,4GlcNAc but not those with the terminal Siaalpha2,3Galbeta1,4GlcNAc. We propose that glycoproteins bearing terminals Siaalpha2,6GalNAc and Siaalpha2,6Gal are endogenous ligands for the ASGP-R, and that the ASGP-R helps to regulate the relative concentration of serum glycoproteins bearing alpha2,6-linked Sia.
Assuntos
Acetilgalactosamina/farmacocinética , Receptor de Asialoglicoproteína/sangue , Glicoconjugados/farmacocinética , Ácido N-Acetilneuramínico/farmacocinética , Acetilgalactosamina/sangue , Acetilgalactosamina/química , Animais , Receptor de Asialoglicoproteína/deficiência , Receptor de Asialoglicoproteína/genética , Ligação Competitiva/genética , Sequência de Carboidratos , Galactose/sangue , Galactose/farmacocinética , Glicoconjugados/sangue , Glicoconjugados/química , Ligantes , Taxa de Depuração Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/sangue , Ácido N-Acetilneuramínico/química , Ligação Proteica/genética , Estrutura Terciária de Proteína , Ratos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) alpha2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Siaalpha2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin and GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the alpha2 helix that is distant from the binding site contribute to the specificity for terminal Siaalpha2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.
Assuntos
Receptor de Asialoglicoproteína/química , Carboidratos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Galinhas , Humanos , Lectinas/metabolismo , Fígado/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Ratos , Proteínas Recombinantes/química , Especificidade da Espécie , SuínosRESUMO
The asialoglycoprotein-receptor (ASGP-R) located on liver parenchymal cells was originally identified and characterized on the basis of its ability to bind glycoproteins bearing terminal galactose (Gal) or N-acetylgalactosamine (GalNAc); however, endogenous ligands for the ASGP-R have not to date been definitively identified. We have determined that the rat ASGP-R specifically binds oligosaccharides terminating with the sequence Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man. Bovine serum albumin chemically modified with 10-15 tetrasaccharides with the sequence Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man is cleared from the blood of the rat with a half-life of <1 min by a receptor located in the liver. We have isolated the receptor and identified it as the ASGP-R. Furthermore, we have determined that subunit 1 of the ASGP-R accounts for the binding of terminal Siaalpha2,6GalNAcbeta. Based on the newly defined specificity of the rat ASGP-R we hypothesize that glycoproteins bearing structures that are selectively modified with terminal Siaalpha2,6GalNAcbeta and are released into the blood may be endogenous ligands for the rat ASGP-R.