An intrinsic mechanism of secreted protein aging and turnover Won Ho Yanga,b, Peter V. Aziza,b, Douglas M. Heithoffc, Michael J. Mahanc, Jeffrey W. Smithb, and Jamey D. Martha,b,c,1 aCenter for Nanomedicine, University of California, Santa Barbara, CA 93106; bSanford–Burnham–Prebys Medical Discovery Institute, La Jolla, CA 92037; and cDepartment of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106 Edited by Kevin P. Campbell, University of Iowa Carver College of Medicine, Iowa City, IA, and approved September 23, 2015 (received for review August 4, 2015) The composition and functions of the secreted proteome are con- activity levels that change in disease (9). We investigated whether trolled by the life spans of different proteins. However, unlike the normal activities of circulating glycosidases may hydrolyze intracellular protein fate, intrinsic factors determining secreted pro- N-glycan linkages attached to secreted proteins, thereby generat- tein aging and turnover have not been identified and characterized. ing multivalent ligands of endocytic lectin receptors in contribut- Almost all secreted proteins are posttranslationally modified with the ing to a mechanism of secreted protein aging and turnover. covalent attachment of N-glycans. We have discovered an intrinsic Results mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-Glycan Remodeling in the Aging of Secreted Proteins. Most se- N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), creted proteins are modified with N-glycans before entry into cir- neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosamini- culatory systems. We isolated secreted proteins among platelet-poor dase B (HexB), possess hydrolytic activities that temporally remodel plasma following i.v. biotinylation and examined them 24, 48, and 72 N-glycan structures, progressively exposing different saccharides h later. Biotinylated secreted proteins were isolated using non- denaturing conditions by affinity chromatography, first with avidin with increased protein age. Subsequently, endocytic lectins with and subsequently with analytical lectins of binding specificities distinct binding specificities, including the Ashwell–Morell receptor, α for Gal, GlcNAc, or Man linkages. Proteomic analyses of plasma integrin M, and macrophage mannose receptor, are engaged in identified 603 noncytosolic proteins bearing lectin ligands consisting N-glycan ligand recognition and the turnover of secreted pro- of Gal linkages, which constituted 99% of the protein mass isolated teins. Glycosidase inhibition and lectin deficiencies increased pro- (Table S1). This diversity further represents the majority of the tein life spans and abundance, and the basal rate of N-glycan plasma proteome identified (10, 11). At steady state in healthy remodeling varied among distinct proteins, accounting for differ- WT mice, the fraction of secreted proteins in plasma bearing Gal ences in their life spans. This intrinsic multifactorial mechanism ligands averaged 2%. We further detected N-glycan remodeling of secreted protein aging and turnover contributes to health and during secreted protein aging with the unmasking of Gal, GlcNAc, the outcomes of disease. and Man linkages (Fig. 1 and Fig. S1). The partial reduction in Sia linkages measured in this time frame indicated that not all glycosylation | protein | homeostasis | glycosidase | lectin N-glycan branches were remodeled. In contrast, Core 1 O-glycans of secreted proteins were not appreciably altered. These data were he secreted proteome is a diverse repertoire of proteins, each consistent with the isolation of different glycoforms of secreted pro- of which is maintained at concentrations appropriate to control teins bearing increasingly remodeled N-glycans with advanced age. T The unmasking of Gal linkages implied the presence of endog- various physiological processes. Although an intrinsic mechanism that enous neuraminidase activity that hydrolyzed Sia linkages. Using determines the life span and turnover of secreted proteins has not antibodies to the four mammalian neuraminidases (Neu1–Neu4) been identified, its existence would explain how the composition of the secreted proteome is regulated and how it might differ in health and disease. Almost all secreted proteins are posttranslationally Significance modified during their biosynthesis and transit through the secretory pathway with one or more N-glycans (1, 2). The N-glycans of mam- In the blood, secreted proteins have different life spans that malian proteins are multiantennary structures consisting of two determine their abundance and function. Measurements of or more oligosaccharide branches that typically include a char- plasma protein composition and biological activities remain acteristic terminal sequence of saccharides. This sequence in- important for many clinical diagnoses. However the molecular cludes sialic acid (Sia) as the distal linkage attached to underlying mechanisms by which secreted proteins age and turnover have galactose (Gal), followed by N-acetylglucosamine (GlcNAc) and remained unidentified. The findings of this research have mannose (Man). Each is present in a multivalent context due to established an intrinsic and constitutive mechanism of secreted N-glycan branching emanating from the core structure. Distinct protein aging and turnover. This mechanism involves multiple secreted proteins are therefore modified with similar N-glycans, factors including circulating glycosidases that progressively each of which includes cryptic ligands of lectins that bind Gal, remodel the N-glycan linkages attached to most secreted pro- GlcNAc, or Man linkages. teins. N-glycan remodeling with time exposes glycan ligands of N-glycans have been primarily perceived as static posttranslational various endocytic lectin receptors that then eliminate these modifications that are eventually degraded along with the attached aged secreted proteins. This mechanism thereby determines protein in the lysosome of the cell. Alterations of N-glycan structures the life spans and abundance of secreted proteins, and mod- thus far identified during a protein’s life span reflect the activity of ulates the pathogenesis and outcomes of disease. exogenous glycosidases or targeted genetic mutations in N-glycan biosynthesis. For example, viral and bacterial neuraminidases cleave Author contributions: J.D.M. designed research; W.H.Y., P.V.A., and D.M.H. performed Sia linkages from the host glycans, thereby contributing to infection research; W.H.Y., M.J.M., J.W.S., and J.D.M. analyzed data; and W.H.Y. and J.D.M. wrote and modulating the severity of disease (3–6). Similarly, exogenous the paper. neuraminidase treatment or a genetic deficiency of the ST3Gal4 The authors declare no conflict of interest. sialyltransferase was found to unmask Gal ligands of lectins known This article is a PNAS Direct Submission. as asialoglycoprotein receptors in reducing the life spans of some 1To whom correspondence should be addressed. Email: [email protected]. secreted proteins (7, 8). It has been noted that blood plasma con- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MEDICAL SCIENCES tains multiple unidentified glycosidases of unknown functions with 1073/pnas.1515464112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1515464112 PNAS | November 3, 2015 | vol. 112 | no. 44 | 13657–13662 Downloaded by guest on September 26, 2021 by Neu inhibition (Fig. 3 F and G and Fig. S4). These findings demonstrated a connection between N-glycan remodeling in secreted protein aging and the rate of protein turnover in cir- culation, implying the presence of one or more endocytic lectin receptors that participate in determining the t1/2s and abun- dance of secreted proteins. Asialoglycoprotein Receptors in the Turnover of Aged Secreted Proteins. The Ashwell–Morell receptor (AMR) of hepatocytes is an endo- cytic lectin discovered by its rapid ability to eliminate from the Fig. 1. N-glycan remodeling of aging secreted proteins. Blood plasma proteins circulation i.v.-administered glycoproteins that have been desialy- from WT mice were isolated at indicated times following i.v. in vivo biotinylation, lated in vitro by Neu activity (7). The AMR binds exposed Gal first by avidin affinity chromatography and next by lectin chromatography. linkages with increased avidity in multivalent contexts, as exist Equivalent amounts of plasma proteins isolated after both chromatographic among the multiple branches of N-glycans (7, 16, 17). AMR steps were compared by SDS/PAGE and lectin blotting. Measurements of lectin function has been found to eliminate desialylated platelets pro- binding were calculated relative to time 0 (60 min following i.v. biotinylation). duced during pneumococcal sepsis and following platelet exposure Lectin binding specificities for glycan linkages are denoted and were confirmed to reduced temperatures (6, 18, 19). in parallel studies with competitive inhibitors (Fig. S1). Data are representative of Among mice lacking either the Asgr1 or Asgr2 protein subunit results from six separate littermate cohort comparisons, and are presented as of the AMR, an accumulation of asialoglycoproteins was detected means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05). ECA, Erythrina cristagalli; among secreted proteins by lectin affinity chromatography using GSL-II, Griffonia simplicifolia-II;