RESUMO
PURPOSE: Wisteria floribunda agglutinin positive human Mac-2-binding protein (WFA(+)-hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin-antibody sandwich immunoassay. In this study, we supplied recombinant WFA(+)-hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA(+)-hM2BP quantification. EXPERIMENTAL DESIGN: The optimum conditions for producing recombinant WFA(+)-hM2BP were selected by cell glycome analysis based on a lectin microarray. Interlot variability of recombinant WFA(+)-hM2BP was determined using an antibody-overlay lectin microarray. Screening of anti-M2BP mAb was completed by incorporating a WFA-antibody sandwich ELISA and an antibody-overlay lectin microarray. RESULTS: The lectin microarray analysis revealed that human embryonic kidney 293 cells efficiently and stably produced WFA(+)-hM2BP in DMEM containing 10% FCS without any variation in the M2BP glycosylation level. A spiking experiment with recombinant WFA(+)-hM2BP was mostly effective for antibody screening. The reconstituted sandwich immunoassay was useful for the continuous quantification and cutoff index expression of serum WFA(+)-hM2BP. CONCLUSIONS AND CLINICAL RELEVANCE: The multiple use of lectin-assisted glycan profiling enabled us to construct a reliable sandwich assay kit for monitoring liver fibrosis in patients with viral hepatitis. This will assist in the development pipeline for other glycodiagnostic agents.
Assuntos
Antígenos de Neoplasias/metabolismo , Diagnóstico , Glicômica/métodos , Glicoproteínas de Membrana/metabolismo , Lectinas de Plantas/metabolismo , Polissacarídeos/metabolismo , Receptores de N-Acetilglucosamina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , HumanosRESUMO
Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (V(max)) of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRP-associated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.