Glycosylation is crucial for a proper catalytic site organization in human glucocerebrosidase

Abstract

Gaucher disease, an autosomal recessive disorder, is caused by a deficiency of glucocerebrosidase (GCase) enzyme, a peripheral membrane-associated glycoprotein that hydrolyses glucosylceramide in lysosomes. Glycosylation is essential for the development of a catalytically active enzyme, specifically in the first site, located at Asn19. However, both the molecular basis of the relevance of N-glycosylation over GCase activity and the effects of glycosylation over its structure and dynamics are still not fully understood. Thus, the present work evaluated GCase enzyme in increasing glycosylation content using triplicate unbiased molecular dynamics simulations. Accordingly, the N-linked glycan chains caused local conformational stabilization effects over the protein, as well as in regions flanking the enzyme catalytic dyad. In the case of the Asn19-linked glycan, it also occurred around region 438-444, where one of the most prevalent GCase mutations is found. Markedly, an increasing catalytic dyad organization was related to increasing glycosylation contents, offering the first atomic-level explanation for the experimental observation that GCase activity is controlled by glycosylation, especially at Asn19.

Insights into the structural biology of Gaucher disease

“…No major conformational changes have been observed in different GCase glycoforms with varied glycosylation content and enzymatic activity remains similar across these glycoforms (Pol-Fachin et al, 2016) and when comparing the three commercially available recombinant GCase enzymes (taliglucerase alpha, imiglucerase and velaglucerase alpha) (Tekoah et al, 2013). As these varied mannose chain lengths and different degrees of glycosylation site occupancy do not affect catalytic activity it indicates that glycosylation is instead responsible for influencing GCase structure, stability and flexibility.…”

“…Presence of Asn 19 glycosylation stabilises regions 438-445, which is the region where a common GD mutation (L444P) occurs. Occupation at this site also lowers the RMSD (root-mean-squared deviation) values for Glu 235 and Glu 340 (Pol-Fachin et al, 2016), suggesting they are in closer proximity, perhaps improving the stability of the catalytic dyad. Glycosylation at the site of Asn 59 increases stability in residues 49-69 and reduces motility at residues 136-156.…”

“…Moreover, the secretion of GCase is a glycosylation-dependant process (Leonova and Grabowski, 2000). N-glycosylation generally confers structural stability of the glycoprotein to which they are attached, increasing plasma residence time and providing steric protection from non-specific interactions and proteases (Pol-Fachin et al, 2016). In fact, the development of a catalytically active GCase enzyme is dependent upon Nglycosylation (Grace and Grabowski, 1990), with occupancy of Asn 19 required for proper functionality of the catalytic dyad (Berg-Fussman et al, 1993).…”

“…There is also reduced flexibility in the region around Asn 270 when occupied, however this is only significantly when the other glycosylation sites are occupied (Pol-Fachin et al, 2016).…”

Produção e purificação da glicocerebrosidase humana recombinante expressa por célula CHO em meio livre de soro fetal bovino

“…NOVO e colaboradores 2012 GSTRAUNTHALER, 2003;BRUNNER et al, 2010 , 2000). O perfil diferenciado da porção oligossacarídica da GCR não afeta a sua estabilidade ou atividade enzimática, porém a sua ausência leva à produção da GCR em sua forma inativa (BERG-FUSSMAN et al, 1993;TEKOAH et al, 2013), pois a glicosilação é crucial para a organização do seu sítio catalítico (POL-FACHIN et al,2016).…”