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. 2015 Nov 3;112(44):13657-62.
doi: 10.1073/pnas.1515464112. Epub 2015 Oct 21.

An intrinsic mechanism of secreted protein aging and turnover

Won Ho Yang  1 , Peter V Aziz  1 , Douglas M Heithoff  2 , Michael J Mahan  2 , Jeffrey W Smith  3 , Jamey D Marth  4
Affiliations

An intrinsic mechanism of secreted protein aging and turnover

Won Ho Yang et al. Proc Natl Acad Sci U S A. .

Abstract

The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell-Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease.

Keywords: glycosidase; glycosylation; homeostasis; lectin; protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
N-glycan remodeling of aging secreted proteins. Blood plasma proteins from WT mice were isolated at indicated times following i.v. in vivo biotinylation, first by avidin affinity chromatography and next by lectin chromatography. Equivalent amounts of plasma proteins isolated after both chromatographic steps were compared by SDS/PAGE and lectin blotting. Measurements of lectin binding were calculated relative to time 0 (60 min following i.v. biotinylation). Lectin binding specificities for glycan linkages are denoted and were confirmed in parallel studies with competitive inhibitors (Fig. S1). Data are representative of results from six separate littermate cohort comparisons, and are presented as means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05). ECA, Erythrina cristagalli; GSL-II, Griffonia simplicifolia-II; LCA, Lens culinaris; MAL-II, Maackia amurensis-II; PNA, peanut agglutinin; RCA, Ricinus communis agglutinin-I, SNA, Sambucus nigra, WGA, wheat germ agglutinin.
Fig. S1.
Fig. S1.
Accumulation of asialoglycoproteins, agalactoglycoproteins, and Man-terminated N-glycans during blood protein aging. (A) Protein biotinylation was performed first in vivo among WT C57BL/6J mice using i.v. injection of biotin, followed by biotinylated protein isolation at multiple times using avidin chromatography. Time 0 is defined as 60 min postbiotin injection, when the in vivo biotinylation of proteins reached maximum and no further biotinylation was detected. Identical amounts (20 μg) of plasma proteins isolated at indicated times postbiotin injection were analyzed by SDS/PAGE and visualized by total protein staining with silver (Left) or by streptavidin binding (Middle). (Right) Total and biotinylated protein concentrations were plotted at the indicated times after in vivo biotinylation. (B) Biotinylated blood plasma proteins from A isolated at the indicated times following i.v. in vivo biotinylation and lectin affinity chromatography were analyzed by lectin blotting (Upper); silver staining (Middle); and by the addition during blotting of 0.2 M Sia, Gal, GlcNAc, or Man (i.e., the relevant glycan competitor) for confirming lectin specificity (Bottom). M.W., molecular weight.
Fig. 2.
Fig. 2.
Identification of circulating neuraminidases and linkage of Neu activity to N-glycan remodeling of aged secreted proteins. (A) Antibodies specific to Neu1, Neu2, Neu3, or Neu4 were used to detect Neu protein abundance in the plasma of mice of indicated strains by ELISA and SDS/PAGE, followed by immunoblotting. M.W., molecular weight. (B) Neu activity measured in mouse plasma. U/L, units per liter. (C) Neu protein abundance in the plasma of healthy human adults by ELISA and SDS/PAGE followed by immunoblotting. (D) Human Neu activity in plasma. (E) Inhibition of Neu activity in plasma following i.v. injection of DANA or zanamivir (250 mg/kg), compared with saline (PBS), administered at time 0 and every 6 h (arrows) over a 24-h period. (F) Biotinylated plasma proteins from WT C57BL/6J mice were isolated at indicated times by lectin affinity chromatography in the presence of PBS or DANA administered every 6 h. Identical amounts of protein (20 μg) were analyzed by SDS/PAGE and lectin blotting. (G) Quantification of lectin ligands detected among plasma proteins in F was normalized to time = 0. Data are representative of results from four to eight separate mouse littermate cohort comparisons and seven human volunteers, and are presented as means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05).
Fig. S2.
Fig. S2.
Pharmacological inhibition of plasma neuraminidase (Neu) activity. (Left) Neuraminidase inhibitor DANA (A) or zanamivir (B) was i.v. injected into WT C57BL/6J mice as the indicated doses and plasma neuraminidase activity were measured after 6 h of DANA or zanamivir injection. (Right) DANA (250 mg/kg) or zanamivir (250 mg/kg) was injected, and plasma neuraminidase activity was measured at the indicated time points. Data are representative of results from four to six littermate cohort comparisons, and are presented as means ± SEM. U/L, units per liter.
Fig. S3.
Fig. S3.
Zanamivir inhibition of N-glycan remodeling during plasma protein aging. (A) Biotinylated blood plasma proteins from WT C57BL/6J mice were isolated at the indicated times by lectin affinity chromatography in the presence of PBS or zanamivir (250 mg/kg) administered every 6 h. Identical amounts of proteins were analyzed by SDS/PAGE and lectin blotting (Top) or total protein staining with silver (Bottom). (B) Measurements of multivalent glycan linkages by lectin binding following administration of PBS or zanamivir calculated relative to time 0. Data are representative of results from four to six littermate cohort comparisons, and are presented as means ± SEM (**P < 0.01; *P < 0.05).
Fig. 3.
Fig. 3.
Alkaline phosphatase t1/2s linked rates of N-glycan remodeling. (A–C) Plasma alkaline phosphatase activity, TNAP abundance, and IAP abundance among WT C57BL/6J mice following administration of PBS, DANA, or zanamivir every 6 h. Circulating t1/2 analyses of TNAP (D) and IAP (E) biotinylated in vivo in the presence of either DANA or PBS administered every 6 h and quantified following immunoprecipitation relative to time 0. Biotinylated TNAP (F) and IAP (G) were isolated from WT C57BL/6J mice at indicated times by lectin affinity chromatography in the presence of PBS or DANA administered every 6 h. Identical amounts of TNAP and IAP were then analyzed by SDS/PAGE, and lectin blotting was quantified normalized to time = 0. Data are representative of results from six to eight separate littermate cohort comparisons, and are presented as means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05). IP, immunoprecipitation.
Fig. S4.
Fig. S4.
TNAP and IAP t1/2s and N-glycan remodeling following zanamivir treatment. (A and B) Expression and t1/2s in blood circulation of biotinylated TNAP and IAP proteins at indicated times in WT mice administered either PBS or zanamivir. (C and D) Lectin blotting analyses from identical amounts of biotinylated, biotin affinity-isolated, and immunoprecipitated TNAP and IAP normalized to time 0 and at indicated times following administration of PBS or zanamivir every 6 h. Data are representative of results from six to eight littermate cohort comparisons, and are presented as means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05). IP, immunoprecipitation.
Fig. 4.
Fig. 4.
AMRs in the turnover of secreted proteins. (A) Plasma proteins from mice lacking Asgr1 or Asgr2 were isolated by lectin affinity chromatography and analyzed by SDS/PAGE and lectin blotting (Top) among amounts of protein indicated by silver staining (Bottom). (B) Quantification of lectin ligands detected among plasma proteins in A normalized to results from WT littermates. (C) Abundance of Neu1, Neu2, Neu3, or Neu4 proteins in plasma. (D) Plasma Neu activity. (E) Plasma TNAP and IAP activities measured in immune precipitates normalized to WT littermates. (F) Abundance of TNAP and IAP proteins in plasma measured by SDS/PAGE and immunoblotting. (G and H) Biotinylated in vivo t1/2 analyses of TNAP and IAP. (I and J) Lectin binding to equivalent amounts of immunoprecipitated TNAP and IAP. Data are representative of six to eight separate littermate cohorts of the indicated genotypes, and are presented as means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05).
Fig. S5.
Fig. S5.
Lectin blotting of total plasma proteins in AMR deficiency. (A) Equivalent amounts of total plasma proteins were analyzed by SDS/PAGE and lectin blotting. (B) Results of lectin blotting in A were quantified relative to results obtained from WT littermates. In separate studies, total blood plasma proteins were subjected to lectin affinity chromatography. (C) Those proteins that did not bind during lectin chromatography (flow-through fraction) were quantified, and equal amounts were analyzed by SDS/PAGE followed by lectin blotting. (D) Results of lectin blotting in C were quantified relative to results obtained from WT littermates. Data are representative of results from four to six separate littermate cohort comparisons, and are presented as means ± SEM.
Fig. S6.
Fig. S6.
Biological processes associated with proteins elevated in AMR deficiency. Proteins elevated in mice lacking either Asgr1 or Asgr2 were subjected to a functional enrichment analysis by gene ontology (GO) terms using the FGnet package in Bioconductor. The 25 top-scoring biological processes are shown.
Fig. S7.
Fig. S7.
TNAP and IAP mRNA abundance in AMR deficiency. (A) Relative TNAP mRNA expression in liver, bone, and kidney tissues from mice of indicated genotypes was measured by quantitative RT-PCR (qRT-PCR). (B) Relative IAP mRNA level from duodenum tissue from mice of indicated genotypes as measured by qRT-PCR. Data are representative of results from six to eight separate littermate cohort comparisons, and are presented as means ± SEM.
Fig. 5.
Fig. 5.
Integrin αM and Mmr in the turnover of secreted proteins remodeled by β-gal and β-N-acetylglucosaminidase activity. (A) Plasma proteins from mice lacking integrin αM (Itgam) were isolated by lectin affinity chromatography and analyzed by SDS/PAGE and lectin blotting. (B) Quantification of lectin ligands detected among plasma proteins in A normalized to WT littermates. (C) β-Gal Glb1 detected in C57BL/6J mouse plasma by immunoblotting. (D) Plasma β-gal activity measured in the absence (PBS) or presence of β-gal inhibitor NN-DGJ (50 mg/kg) administered at time 0 and every 12 h (arrows) over a 72-h period. (E) Quantification of lectin ligands detected among plasma proteins in the presence of PBS or NN-DGJ normalized to time 0. (F) Plasma proteins from mice lacking Mmr were isolated by lectin affinity chromatography and analyzed by SDS/PAGE and lectin blotting. (G) Quantification of lectin ligands detected among plasma proteins in F normalized to WT littermates. (H) β-N-acetylglucosaminidase (β-GlcNAcase) HexB detected in C57BL/6J mouse plasma by immunoblotting. (I) Plasma β-N-acetylglucosaminidase activity measured in the absence (PBS) or presence of β-N-acetylglucosaminidase inhibitor 2-acetoamido-1,2-dideoxynojirimycin (2-ADN; 25 mg/kg) administered at time 0 and every 12 h (arrows) over a 72-h period. (J) Quantification of lectin ligands detected among plasma proteins in the presence of PBS or 2-ADN normalized to time 0. Data are representative of six to eight separate littermate cohorts of the indicated genotypes, and are presented as means ± SEM (***P < 0.001; **P < 0.01; *P < 0.05).
Fig. S8.
Fig. S8.
Pharmacological inhibition of plasma β-gal activity and β-GlcNAcase activity in secreted protein abundance and turnover. (A) Inhibition of β-gal activity following i.v. administration of NN-DGJ with varying dosage and measurements of activity at multiple times after a single administration. (B) β-Gal inhibitor NN-DGJ was i.v. injected into WT C57BL/6J mice (50 mg/kg) every 12 h. Identical amounts of biotinylated glycoproteins isolated from plasma using lectin affinity chromatography were separated by SDS/PAGE and analyzed by lectin binding (Left) or by protein staining with silver (Right). (C) Inhibition of β-GlcNAcase activity following i.v. administration of ADN with varying dosage and measurements of activity at multiple times after single administration. (D) β-GlcNAcase inhibitor ADN was i.v. injected into WT C57BL/6J mice (25 mg/kg) every 12 h. Identical amounts of biotinylated glycoproteins isolated from plasma using lectin affinity chromatography were separated by SDS/PAGE and analyzed by lectin binding (Left) or by protein staining with silver (Right). Data are representative of results from four to six littermate cohort comparisons, and are presented as means ± SEM.
Fig. 6.
Fig. 6.
An intrinsic mechanism of secreted protein aging and turnover. Multiantennary N-glycans of secreted proteins are progressively hydrolyzed by exoglycosidases, including neuraminidase (also known as sialidase), galactosidase, and glucosaminidase, during protein aging, generating remodeled N-glycans with unmasked multivalent Gal, GlcNAc, and Man linkages. These remodeled N-glycans are ligands of various endocytic lectin receptors. Multiple glycosidases and lectin receptors together determine secreted protein life span and abundance.
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