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LC-MS/MS analysis of the cation-independent mannose 6-phosphate receptor (CI-MPR) from bovine serum

ABSTRACT: The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. To evaluate the allosteric properties and conformation of CI-MPR, we isolated the receptor from a mammalian source, bovine serum and analyzed the sequence of the isolated receptor by mass spectrometry.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Blood Serum

SUBMITTER: Mayumi Ishihara-Aoki

LAB HEAD: Kazuhiro Aoki

PROVIDER: PXD052183 | Pride | 2024-05-20

REPOSITORIES: Pride

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			Action	DRS
	2153-Porter_220811_Ts024_CIMPR_01.mzML	Mzml
	2153-Porter_220811_Ts024_CIMPR_01.mzid.gz	Mzid
	2153-Porter_220811_Ts024_CIMPR_01.raw	Raw
	2153-Porter_220811_Ts024_CIMPR_02.raw	Raw
	2165-Porter_230104_Ts024_11-4-22_01.mzML	Mzml

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Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.

Dataset Information

LC-MS/MS analysis of the cation-independent mannose 6-phosphate receptor (CI-MPR) from bovine serum

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