Domain 5 of the Cation-Independent Mannose 6-Phosphate Receptor Preferentially Binds Phosphodiesters (Mannose 6-Phosphate N-Acetylglucosamine Ester)

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• 作者：Carrie A. Chavez ; Richard N. Bohnsack ; Mariko Kudo ; Russell R. Gotschall ; William M. Canfield ; Nancy M. Dahms
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 6, 2007
• 年：2007
• 卷：46
• 期：44
• 页码：12604 - 12617
• 全文大小：696K
• 年卷期：v.46,no.44(November 6, 2007)
• ISSN：1520-4995

文摘

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDacation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system thatbind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them fromthe secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmicregion. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues(Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containingsingle amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) arecritical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydratebinding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) orphosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated bytreating the lysosomal enzyme acid -glucosidase (GAA) with recombinant GlcNAc-phosphotransferaseand uncovering enzyme (N-acetylglucosamine-1-phosphodiester -N-acetylglucosaminidase). SPR analysesusing these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of thereceptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.