Supplemental Material can be found at: http://www.mcponline.org/cgi/content/full/M600030-MCP200 /DC1 Research Identification and Validation of Mannose 6-Phosphate Glycoproteins in Human Plasma Reveal a Wide Range of Lysosomal and Non-lysosomal Proteins*□S David E. Sleat‡§¶, Yanhong Wang‡, Istvan Sohar‡, Henry Lackland‡, Yan Liʈ, Hong Liʈ, Haiyan Zheng‡, and Peter Lobel‡§** Acid hydrolase activities are normally confined within the potential utility as biomarkers. Molecular & Cellular cell to the lysosome, a membrane-delimited cytoplasmic Proteomics 5:1942–1956, 2006. organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain The lysosome is an acidic, membrane-delimited organelle mannose 6-phosphate (Man-6-P), a modification on N- Downloaded from that is responsible for the degradation and recycling of mac- linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins romolecules, playing a role in endocytosis and autophagy (1). to the lysosome. In this study, we purified the Man-6-P Most of the resident hydrolases (e.g. proteases, glycosidases, glycoforms of proteins from human plasma by affinity lipases, phosphatases, sulfatases, and nucleases) and acces- chromatography on immobilized MPRs and characterized sory proteins (e.g. activator proteins) are transported to the this subproteome by two-dimensional gel electrophoresis lysosome by the mannose 6-phosphate (Man-6-P)1 targeting www.mcponline.org and by tandem mass spectrometry. As expected, we iden- pathway. Like many other glycoproteins, soluble lysosomal tified many known and potential candidate lysosomal pro- proteins are synthesized in the endoplasmic reticulum and are teins. In addition, we also identified a number of abundant cotranslationally glycosylated on select asparagine residues. classical plasma proteins that were retained even after As these proteins move through the secretory pathway, the two consecutive rounds of affinity purification. Given their lysosomal proteins are selectively recognized by a phospho- abundance in plasma, we initially considered these pro- by on July 11, 2008 transferase that initiates a two-step reaction that results in the teins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified generation of the Man-6-P modification on specific N-linked glycopeptides revealed that some proportion of these oligosaccharides. The modified proteins are then recognized classical plasma proteins contained the Man-6-P modifi- by two Man-6-P receptors (MPRs), the cation-dependent MPR cation. We propose that these glycoproteins are phos- and the cation-independent (CI-) MPR (2). These receptors bind phorylated at low levels by the lysosomal enzyme phos- the phosphorylated lysosomal proteins in the neutral environ- photransferase, but their high abundance results in ment of the trans-Golgi network and travel to an acidic prely- detection of Man-6-P glycoforms in plasma. These results sosomal compartment in which the low pH promotes dissoci- may provide useful insights into the molecular processes ation of the receptors and ligands. The receptors then recycle underlying Man-6-phosphorylation and highlight circum- back to the Golgi to repeat the process or to the plasma mem- stances under which the presence of Man-6-P may not be brane. Here the CI-MPR can function in the endocytosis and indicative of lysosomal function. In addition, characteriza- lysosomal targeting of extracellular Man-6-P glycoproteins. tion of the plasma Man-6-P glycoproteome should facili- tate development of mass spectrometry-based tools for The primary location and site of function of lysosomal en- the diagnosis of lysosomal storage diseases and for in- zymes is by definition intracellular. However, lysosomal activ- vestigating the involvement of Man-6-P-containing glyco- ities have also been identified in a variety of extracellular proteins in more widespread human diseases and their 1 The abbreviations used are: Man-6-P, mannose 6-phosphate; MPR, Man-6-P receptor; CI-MPR, cation-independent MPR; sCI- From the ‡Center for Advanced Biotechnology and Medicine and MPR, soluble cation-independent MPR; HS, Heremans-Schmid; bis- §Department of Pharmacology, University of Medicine and Dentistry Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; of New Jersey, Piscataway, New Jersey 08854 and the ʈDepartment 1D, one-dimensional; 2D, two-dimensional; GPM, Global Proteome of Biochemistry and Molecular Biology, New Jersey Medical School, Machine; BTD, biotinidase; PTGDS, prostaglandin-H2 D-isomerase; Newark, New Jersey 01701-1709 CP, ceruloplasmin; CPVL, carboxypeptidase vitellogenic-like; Received, January 23, 2006, and in revised form, April 20, 2006 SMPDL3A, acid sphingomyelinase-like protein 3a; CREG1, cellular Published, MCP Papers in Press, May 17, 2006, DOI 10.1074/ repressor of E1A-regulated gene transcription; EPDR1, mammalian mcp.M600030-MCP200 ependymin-related protein. 1942 Molecular & Cellular Proteomics 5.10 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org The Human Plasma Mannose 6-Phosphate Glycoproteome environments including plasma (3). These proteins may be Man-6-P or lysosomal function. Many of these proteins are delivered to the plasma through leakage from dead or dying highly abundant plasma glycoproteins that we initially sus- cells, through mobilization of secretory lysosome or granule pected to represent likely contaminants that do not contain contents, or by the release of lysosomal residual bodies by Man-6-P. Therefore, to distinguish between true Man-6-P cell defecation. It is also possible that a portion of the newly glycoproteins and contaminants, we used a technique to di- synthesized lysosomal proteins escape the intracellular tar- rectly identify Man-6-P-modified glycopeptides. Unexpect- geting pathway for transport to the lysosome and are se- edly we found that a small proportion of multiple abundant creted. Evidence for the latter is well documented; for exam- classical plasma proteins exist as Man-6-P-containing ple, in cultured mouse embryonic fibroblasts, wild-type cells glycoforms. secrete a small proportion of Man-6-P glycoproteins (ϳ10% of that secreted in the absence of both MPRs, depending on EXPERIMENTAL PROCEDURES the protein) (4–7), indicating that the sorting of newly synthe- Purification of Human Plasma Man-6-P Glycoproteins—Refrozen sized lysosomal proteins is not absolutely efficient. lots of thawed human plasma were generously provided by V. I. The biological significance of circulating lysosomal proteins Technologies, Inc. (Melville, NY). Plasma Man-6-P glycoproteins were is unclear, but there are several possibilities. First, some hy- purified using a modification of the affinity protocol described for human brain (17). For each purification, ϳ2 liters of frozen human drolases may be synthesized and released by one cell type plasma were rapidly thawed at 37 °C and then clarified by centrifu- and delivered to another, and so circulating lysosomal proteins gation at 13,000 ϫ g for 90 min at 4 °C. The supernatant was filtered may represent intermediates in this process. Second, circulat- through six layers of cheesecloth and then added to an equal volume ing lysosomal proteins might have a specific function in plasma. of ice-cold PBS containing 10 mM ␤-glycerophosphate, 10 mM EDTA, Downloaded from Third, the presence of lysosomal proteins in plasma may simply 2% Triton X-100, 0.4% Tween 20, and 0.2 mM Pefabloc (Pentafarm, Basel, Switzerland). The diluted plasma was then applied to a column represent the steady state levels reflecting the balance between of immobilized pentamannosyl phosphate-aminoethyl agarose the rate of unwanted appearance in plasma (due to leakage (100-ml bed volume) to remove circulating soluble cation-indepen- and/or lack of absolute fidelity in the intracellular targeting sys- dent MPR (sCI-MPR), and the flow-through was applied to a column tem) versus the rate of uptake and clearance. of immobilized sCI-MPR (100-ml bed volume at a coupling density of www.mcponline.org Regardless of biological function, circulating lysosomal hy- 3.3 mg/ml) at a flow rate of ϳ200 ml/h to capture circulating Man-6-P glycoproteins. The MPR resin was batch washed with 3 ϫ 1 column drolases potentially represent valuable biomarkers for the volume of PBS containing 5 mM EDTA, 5 mM ␤-glycerophosphate, 1% study and diagnosis of human diseases. Mutations in genes Triton X-100 and then with 3 ϫ 1 column volume of the same buffer encoding lysosomal proteins result in over 40 storage dis- without Triton X-100. The column was flow-washed with 20 column eases (for a review, see Ref. 8). In addition, lysosomal activ- volumes of PBS/EDTA/␤-glycerophosphate at 125 ml/h and then ities have also been indirectly implicated in more widespread eluted with 2 column volumes of the same buffer containing 10 mM by on July 11, 2008 pathogenic processes (9–12) including tumor invasion and Man-6-P at a flow rate of 100 ml/h. Fractions containing Man-6-P glycoproteins were identified by protein and ␤-mannosidase assay, metastasis, Alzheimer disease, rheumatoid arthritis, and ath- pooled, concentrated by ultrafiltration (Centriprep-10, Millipore, Bil- erosclerosis as well as in normal processes such as aging (13) lerica, MA), and buffer-exchanged to either 100 mM ammonium bi- and immune system function (14). As a class, lysosomal pro- carbonate (single purification protocol) or PBS/EDTA/␤-glycerophos- teins represent a relatively small subgroup of the plasma phate (first elution from the double purification protocol). For proteome that should be amenable to quantitative analysis. repurification, the buffer-exchanged Man-6-P eluate was reapplied to the column of immobilized MPR and purified essentially as described In this study, we set out to characterize the human plasma above. The Man-6-P eluate was buffer-exchanged with 100 mM am- proteome of mannose 6-phosphate glycoproteins to provide monium bicarbonate.