Glyco

PMCID: PMC3975653

Legend: Gene, Sites, Suger

Section : HDL particles remain poorly characterized due to their size, complexity,dynamic nature, and large degree of diversity in com position and function

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Enabling technologies that can be used to comprehensively analyzethese biologically important particles are needed
In particular,very little is known about the biological functions related to theglycosylated components of HDL
This deficiency in understanding theglycobiology of HDL may be critically important since early publishedevidence indicates that both glycoproteins and glycolipids associatedwith HDL and their variation in glycosylation patterns may be eithermechanistically involved or diagnostic of HDL functions
In this study, HDL particles isolated from healthy human plasma were comprehensivelyexamined for their glycan structures, and the results led to a compellingnew model of HDL as a class of highly sialylated particles

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Section : ProteomicsAnalysis

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Tryptic peptides were digestedfrom HDL fractions isolated by ultracentrifugation
After high-performanceliquid chromatography (HPLC) separation via reversed phase C18 column,the peptides were analyzed by tandem MS. Following a database search,17 proteins were identified shown in SupplementalTable S1, Supporting Information from one replicate as an example
The base −10 log of the expectation that any particular proteinassignment was made at random was lower than −2
ApoA-I wasobserved as the major protein
In humans, about 60% of the proteincontent in HDL is represented by ApoA-I
Other apoproteins were present, including Apo C-II, C-III, D, E,and M, which are typical minor apoproteins associated with HDL
Onthe other hand, ApoB-100, the major constituent of VLDL and LDL, wasnot present
Other proteins known to be associated with HDL were alsoobserved, including acute phase response proteins such as SAA , liversecreted proteins such as alpha-2-HS-glycoprotein ( fetuin A ), andthe key mediators of the complement system C3 inhibitor
The proteins associated with HDL isolated from healthy human plasmain this study have all been suggested as HDL-associated proteins inprevious studies
These results confirmed our HDL isolation approach and provideda list of proteins for site-specific glycosylation analysis

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Section : Glycansfrom HDL Com position ally Profiled by Nano-LC–MS. TheN-glycans were released using the protocol described in Experimental Procedures

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Analyses were done fromthe isolated HDL triplicates
The glycan profile of HDL is illustratedwith one of the triplicates (Figure 2)
Thedepicted glycan structures were based on biological precedence andtheir correct masses; however, isomers are putative
HPLC-microchip packed with a PGC stationary phase was usedto separate glycans with high sensitivity and reproducibility
The total ion chromatogram (TIC) from the raw LC–MS datawas separated into a number of extracted compound chromatograms (ECCs)
The extractor algorithm was based on the expected charge carriers,potential neutral mass losses, and a predicted isotopic distribution
Each peak represents one compound eluting at a certain retention timewith the detected m/z, which enablesthe assignments of glycan com position
The most abundant glycan (includingisomers) was a biantennarycomplex type glycan with two sialic acids (Hexose5HexNAc4Neu5Ac2)
Perhaps not surprisingly, these glycansare also the most abundant in blood
Inthis regard, HDL-associated proteins are similar to other abundantglycoproteins in blood
Two abundantisomers were observed eluting at retention times 23 and 25 min
Mostof the glycans (90%) from HDL glycoproteins were sialylated with oneor two Neu5Ac(s)
Ion intensities were measured in the MS mode basedon the protonated molecular ion in positive mode (Table 1)
Following the most abundant glycan with two Neu5Ac , a complextype biantennary glycan with one Neu5Ac was shown in a high intensityeluting at 17–20 min including four observed isomers
Our groupreported glycans with sialic acids are retained longer with PGC
Here we also observed that the sole additionof an acidic NeuAcresidue increased the retention time by nearly 5 min
Sialic acid , mostly found as a terminal componentof glycoproteinsand glycolipids on the outer surface of cells, is involved in cellularsecretions
Sialic acid participatesin multiple and diverse cellular events, such as acting as an antirecognitionagent by shielding recognition sites and conversely by beinga biological recognition site as a ligand for multiple molecules
Sialic acid is anionic and therefore likely contributes to HDL ’snegative charge
Previously, it has been shown that the addition ofphosphatidylinositol, which increases HDL ’s negative charge,blocked binding of HDL to hepatic lipase
Other previous studies have also shown that the electronegativityof HDL affects its binding to cholesterol ester transfer protein andthus the exchange of cholesterol esters with LDL
The sialylated glycans in HDL associated proteins may beinvolved in molecular and cellular interactions that affect the functionof HDL
For example, sialic acid residues of ApoE may be essentialfor its recognition by HDL3 particles and further the bindingto HDL3
Interestingly, desialylationof lipoprotein-associated apoproteins was found to be associated withincreased circulating neuraminidase excreted by Streptococcuspneumonia in patients with hemolytic uremic syndrome, suggesting that bacterial infection may leadto desialylation of HDL particles in vivo
In addition to complex-typesialylated N-glycans, fucosylated N-glycanswere present at relatively low abundances
High-mannose and hybridtype N-glycans were also observed, indicating the variety of N-glycansfrom HDL-associated proteins
The wide dynamic range of the detectedN-glycans by the described method allows the evaluation of all typesof oligosaccharides and further analyses of their biological functions
Supplemental Figure S1, Supporting Information shows the overlaid ECCs of N-glycans from the three replicates
The separation of glycans and their profiles was similar, as the mostabundant ones were observed as sialylated complex-type eluting at23–25 min
The method is considered reproducible with regardto the glycan com position assignments from these technical triplicates

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Section : Site-Specific Analysis of HDL Glycoproteins

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Glycopeptideswere assigned based on a combination of the MS/MS data and the accurateprecursor ion mass measurement
The advantage of nonspecific digestionwith Pronase is the ability of the enzyme mixture to cleave the protein(s)into short peptides with typically one glycosylation site
Previousstudies from our group on the analysis of nonspecific digested glycopeptideswith collision-induced dissociation (CID) experiments revealed detailedand comprehensive glycan com position al information for each glycopeptide (both N- and O-linked)
Glycosidic bond cleavages(B- and Y-type ions) were the major products as well as some minorpeptide fragmentations
Table 2 liststhe Pronase-digested glycopeptides from HDL-associated glycoproteinswith their glycan com position, glycosylation site , protein ID, andintensity
The most abundant N-glycan (Hexose5HexNAc4Neu5Ac2) was found in multiple glycoproteins fromthe glycopeptide assignment
Here, all the identified proteins fromthe HDL were confirmed in triplicate experiments using X
Tandem andwere used in the glycopeptide assignment search
A number of deconvolutedglycopeptide MS/MS spectra are shown as examples in Figure 3
Figure 3A represents theMS/MS spectrum for a triprotonated fetuin A glycopeptide containingthis most abundant disialylated N-glycan
Particularly unique to glycopeptidescontaining sialylated glycans are B-type ions corresponding to neutralmass 291 Da (Neu5Ac) and neutral mass 273 Da (Neu5Ac-H2O).
Mass peaks also include those observed as 203 Da, 365 Da, and656 Da, which correspond to the neutral masses of HexNAc, (Hex + HexNAc),and (Hex + HexNAc + Neu5Ac).
Because of the labile nature of sialicacid residues and their positions at the termini , the initial lossof sialic acid was commonly observed with the sialylated glycopeptides
Following the sequential neutral losses of monosaccharides of Neu5Ac,hexose, and HexNAc, the CID data revealed the mass peak 832 Da correspondingto glycopeptide (APLNDT + HexNAc)
The presence of (peptide + HexNAc)is considered a valuable means for validating the assignment of glycopeptideparticularly when analyzing complex protein mixtures
Pronasedigestion simultanously enables site-specific analysisof both N- and O-glycans
In addition to the abundant N-glycans discussedpreviously, a number of O-glycopeptides were present attached withsialylated O-glycans (Table 2)
The observedO-glycans associated with the HDL glycoproteins were all sialylated,comfirming that HDL particles are highly sialylated
Figure 3B,C revealed the deconvoluted MS/MS spectra fortwo diprotonated O-glycopeptides from ApoC-III and fetuin A, respectively
Most O-glycopeptides analyzed in the HDL mixture contained a core1 type with two Neu5Acs
Figure 4 showsthe site heterogeneity offour glycoproteins from eight of the identified HDL associated glycoproteinsas an example
Fetuin A is a multifunctional circulating liver-derivedglycoprotein in serum and plasma
Severalstudies have suggested that fetuin A may be critically important tocardiovascular health
Both N- and O-glycans were observed at multiple glycosylation siteswith fetuin A, all of which were sialylated (Figure 4A), which is consistent with the previous glycosylation analysison the individual protein fetuin A . Themost abundant glycan (Hexose5HexNAc4Neu5Ac2) was disialylated and was found on most of the glycoproteinsexamined
It was present at ASN156 and ASN176 of fetuin A . Angiotensinogen is a heterogeneous glycoprotein mainly producedby hepatocytes in plasma, which has a well-known role in blood pressureregulation in animals and humans
Ithas previously been shown that the glycosylation of angiotensinogenmay play a significant role in its functional heterogeneity
Results of site heterogeneity of angiotensinogenare summarized in Figure 4B
Among the fourpotential N-glycosylation sites , three identified sites were occupiedwith N-glycans in this study
While two sites were observed associatedwith sialylated N-glycans, site ASN304 was attached withfucosylated N-glycans
Angiotensinogen was also found attached withthe most abundant sialylated glycan (Hexose5HexNAc4Neu5Ac2) at ASN170
Figure 4C details the glycan associatedwith alpha-1B-glycoprotein ( A1BG )
A1BG is believed to be a memberof the immunoglobulin family
The monosialylatedglycan (Hexose5HexNAc4Neu5Ac1) attachedat ASN363 on A1BG
The glycosylation of apolipoproteinC3 ( ApoC-III ) was analyzedby a couple of groups, showing the distribution of O-glycans
The disialylated O-glycan at site Thr94 from ApoC-IIIis shown in Figure 4D
ApoC-III inhibits lipoproteinlipase and hepatic lipase , thought to inhibit hepatic uptake of triglyceride-richparticles
ApoC-III was also recentlyfound to be increased in HDL isolated from patients with both stablecoronary artery disease and acute coronary syndrome as well as inhemodialysis patients
In general, for the firsttime the site-specific glycosylationof these proteins in HDL particles has been described
Compared withprevious disscussed glycan analysis, some glycans were not observedin the glycopeptide analysis due to their low abundances as well asthe potential short peptide length from Pronase digestion
However,the functions they impart on HDL through their glycosylation, andwhether alterations in their glycosylation as part of HDL particlesis either causative or diagnostic in disease, are unknown

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Section : HDL GangliosidesCharacterized by MS/MS

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Gangliosidesare another contributor of sialic acid in HDL
We applied a high resolutionquadrupole time of flight mass spectrometry (Q-TOF MS) method withreverse-phase nanoHPLC separation for the analysis of human HDL gangliosides
The high mass accuracy of these instruments, along with the MS/MScapability, made it possible to perform focused detection of gangliosidespecies in the polar lipid extract
Postprocessingprecursor ion scans indicate the existence of gangliosides, whichelute at certain retention times
Moreover, based on accurate mass,glycan information and ceramide com position were simultaneously obtained
Figure 5 is representative of the ECCs of HDL gangliosides
The chromatogram reveals an elution pattern startingwith gangliosides with shorter ceramide chains followed by those withlonger ceramides
Examination of the profiles shows that GM3 (monosialoganglioside,NeuAc2–3Gal1–4Glc–Cer) and GD3 (disialoganglioside,NeuAc2–8NeuAc2–3Gal1–4Glc–Cer) were abundantganglioside ions in HDL from healthy human plasma, in agreement withearlier studies
Eight GM3 and four GD3gangliosides were detected
A 60% GM3 and 40% GD3 distribution wasobserved, and ion intensities were measured in the MS mode based onthe deprotonated molecular ion (Table 3)
Interestingly,the com position of human aorta from patients who had died of myocardialinfarction were found to have higher GD3 content in the aortic mediaversus the intima, and total gangliosidecontent was found to be lower in cells isolated from atheroscleroticcompared with normal human aorta
Gangliosideshave even been found to stimulate lipoxygenase-mediated eicosanoidproduction in peripheral blood lymphocytes, and GD3 was more stimulatorycompared with GM1 or GM3
These previousreports suggest that ganglioside content and com position may haveimportant biological implications in heart disease and its relatedmechanisms involving lipoproteins
The analysis also revealed the com position of themajor ceramideportion of GM3 and GD3 gangliosides
Both GM3 and GD3 were composedof heterogeneous ceramide lipid tails , including d18:1/16:0 and d18:1/23:0
Types of sphingoid bases and fatty acids in the backbones were determinedwith the use of 80 V MS/MS
Fragments at m/z 264.269 and at m/z 236.238indicated the existence of sphingoid bases d18:1 and d16:1, respectively
As an example, GM3 (d18:1/16:0) yields a diagnostic sialic acid fragmentat m/z 290.09 in 40 V negative modeMS/MS, while 80 V positive mode MS/MS spectrum provides the ceramideinformation at m/z 520.49 and 264.27(Supplemental Figure S2, Supporting Information)
The importance of the ceramide lipid tail com position al differencesis currently poorly understood
For example, transmembrane anchoredangiotensin converting enzyme , an enzyme important for blood pressureregulation, was found to associate only with C18 but not C16 sphingomyelin-containingrafts
A recent report showed that nascent HDL resemble lipid rafts, suggesting the possibility that HDL particleshave micro-heterogeneity with lipid raftlike portions of the phospholipidouter layer that recruit specific gangliosides and proteins to performspecific functions, just as occurs in plasma membrane lipid rafts
The simultaneous identification of the glycanand ceramide will provide a clearer picture of the role of gangliosidesin lipoprotein biology

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Section : Overlaid extracted compoundchromatograms (ECCs) showing the profileof human HDL glycans via nano-LC–MS. Green circles, yellowcircles, blue squares, red triangles, and purple diamonds representmannose, galactose, GlcNAc, fucose, and NeuAc residues , respectively

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Section : Deconvoluted MS/MS spectra of three sialylated glycopeptides

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(A)MS/MS data for an N-linked glycopeptide from fetuin A in HDL proteinmixture ; (B and C) MS/MS data for O-linked glycopeptides from apolipoproteinC3 and fetuin A , respectively

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Section : Glycan site-heterogeneity of HDL associated glycoprotein (A) fetuinA ( alpha-2-HS-glycoprotein ), (B) angiotensinogen , (C) alpha-1B-glycoprotein ,and (D) apolipoprotein CIII

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Section : Overlaid extracted compound chromatograms(ECCs) showing the profileof human HDL ganglioside via nano-LC–MS. Analysis of the accuratelymeasured masses corresponding to each peak in chromatograms revealsmonosialylated ganglioside (GM3, peaks shaded in blue) and disialylatedganglioside (GD3, peaks shaded in pink)

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Protein	NCBI ID	SENTENCE INDEX