RESUMO
Congenital disorders of glycosylation (CDG) are genetic diseases characterized by deficient synthesis (CDG type I) and/or abnormal processing (CDG type II) of glycan moieties linked to protein and lipids. The impact of the molecular defects on protein glycosylation and in turn on the clinical phenotypes of patients with CDG is not yet understood. ALG12-CDG is due to deficiency of ALG12 α1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the endoplasmic reticulum. ALG12-CDG is a severe multisystem disease associated with low to deficient serum immunoglobulins and recurrent infections. We thoroughly investigated the glycophenotype in a patient with novel ALG12 variants and immunodeficiency. We analyzed serum native transferrin, as first line test for CDG and we profiled serum IgG and total serum N-glycans by a combination of consolidated (N-glycan analysis by MALDI MS) and innovative mass spectrometry-based protocols, such as GlycoWorks RapiFluor N-glycan analysis coupled with LC-ESI MS. Intact serum transferrin showed, as expected for a CDG type I defect, underoccupancy of N-glycosylation sites. Surprisingly, total serum proteins and IgG N-glycans showed some specific changes, consisting in accumulating amounts of definite high-mannose and hybrid structures. As a whole, ALG12-CDG behaves as a dual CDG (CDG-I and II defects) and it is associated with distinct, abnormal glycosylation of total serum and IgG N-glycans. Glycan profiling of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Deficiência de IgG/genética , Imunoglobulinas/genética , Manosiltransferases/genética , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Glicoproteínas/sangue , Glicosilação , Humanos , Deficiência de IgG/sangue , Deficiência de IgG/metabolismo , Deficiência de IgG/patologia , Imunoglobulinas/sangue , Imunoglobulinas/deficiência , Lactente , Masculino , Manosiltransferases/sangue , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Polissacarídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transferrina/genética , Transferrina/metabolismo , Sequenciamento do ExomaRESUMO
To determine whether abnormal metabolism of L-fucose in hepatocellular carcinoma is accompanied by alterations in the activities of fucosyltransferases, the latter were determined in plasma and liver tissue of patients with this disease and in cirrhotic and normal subjects. Activities of alpha-2/alpha-3 and alpha-6-L-fucosyltransferases were all significantly greater in plasma from patients with hepatocellular carcinoma than in plasma from cirrhotic patients or normal subjects (p less than 0.025). The activity of each enzyme was dependent, to a similar extent, on Mn2+, Mg2+ and triton X-10, irrespective of the source, and all displayed pH optimums in the range of 7.5 to 8.0. In contrast, activities of alpha-2/alpha-3 fucosyltransferases were significantly lower (p less than 0.025) in homogenates prepared from tumorous liver tissue than in that prepared from nontumorous tissue from hepatocellular carcinoma and cirrhotic patients, whereas for the alpha-6 enzyme the situation was reversed (typically, tumor tissue levels were 5 pmol/hr/mg; in nontumor tissue they were 2 pmol/hr/mg). Activities of galactosyl and mannosyltransferase in tumor tissue were greater in all cases than in nontumor cirrhotic tissue. Plasma fucosyltransferases are specifically elevated in hepatocellular carcinoma but different mechanisms appear to underlie the changes seen for alpha-2/alpha-3 and alpha-6-L-fucosyltransferases.
Assuntos
Carcinoma Hepatocelular/enzimologia , Fucosiltransferases/metabolismo , Neoplasias Hepáticas/enzimologia , Adulto , Idoso , Carcinoma Hepatocelular/sangue , Detergentes/farmacologia , Fucosiltransferases/sangue , Galactosiltransferases/sangue , Humanos , Concentração de Íons de Hidrogênio , Íons , Fígado/enzimologia , Neoplasias Hepáticas/sangue , Masculino , Manosiltransferases/sangue , Metais/farmacologia , Pessoa de Meia-Idade , alfa-L-Fucosidase/metabolismoRESUMO
The total sialic acid content of blood platelets from rats raised for 8 weeks or 12 months on a diet containing 1% linoleic acid (1LA) was significantly lower (by over 30%) than that from those raised on an isocalorific diet containing 6% linoleic acid (6LA). The transfer of sialic acid to endogenous glycoprotein acceptor was also significantly lower (up to almost 4-fold) in 1LA platelet and megakaryocyte-rich preparations but the transfer to exogenous glycoprotein acceptor was similar in both 1LA and 6LA platelets. The megakaryocyte-rich fraction of 1LA animals showed a reduced phosphodolichol-sensitive N-acetylglucosaminyl (but not mannosyl) transfer to endogenous glycoprotein compared with 6LA animals. No significant difference was found between the megakaryocytes of 1LA and 6LA animals in the incorporation of radioactive mannose and glucosamine into the glycoprotein of the whole cells. It was concluded that the decreased transfer of sialic acid to glycoproteins of platelets and megakaryocyte of animals on the 1LA diet was due to the decreased availability of sialyl acceptor. The formation of N-linked oligosaccharide was the same in both 1LA and 6LA megakaryocytes, and thus any differences in phosphodolichol-mediated N-glycosylation did not account for this decreased availability of sialyl acceptor.
