Glyco

Title : Our recent analysis of Hp isolated from serum of a patient withHCC detected multiply fucosylated glycans with up to six fucoses

Abstract :

We have used glycosidase assisted LC–MS/MS to show the presenceof Lewis Y type glycoforms at one glycopeptide of Hp
In this study,we use the recently optimized glycosidase-assisted LC–MS/MSmethods and MSn structural analysis of detached N-glycans to compareglycoforms of Hp with glycoforms of Hpx , Kng-1 , and CFH isolated fromplasma of the same patient
Our goal is to compare site-specific microheterogeneityof proteins synthesized by the same enzymatic machinery and underthe same pathophysiological conditions
The proteins were purifiedby reversed-phase chromatography after incubation of the patient’splasma with heme-agarose beads
Isolation of these proteins as a complexon heme-agarose is expected because all three proteins have high affinityfor either heme or each other
BecauseN-glycans have minimal interaction with the reversed-phase resin,all glycoforms of the protein coelute and their microheterogeneitycan be analyzed efficiently in the isolated protein
Hpx and CFH wereboth purified to apparent homogeneity, but Kng-1 coeluted from thereversed-phase column with histidine-rich glycoprotein and other minorcontaminants (Figure 1)
We were able to analyzeglycopeptides of Kng-1 efficiently in the simple coeluting mixtureof proteins , but we were not able to isolate sufficient amounts ofpurified Kng-1 for MSn structural analysis; MSn analysis was thereforecompleted on Hp, CFH , and Hpx
We have focused on LC–MS analysisof tryptic digests because trypsin provides sufficient coverage ofeach protein ’s glycosites to allow comparison of microheterogeneityand because the tryptic digest is standardized sufficiently to allowsemiquantitative comparison of precursor ion intensities of the microheterogeneousglycopeptides
We describe the first results of analysis of each proteinand follow the description by comparison of the results across the proteins
Haptoglobin : Hp is a liver-secreted glycoprotein with fourN-glycosylationsites
Its major function is capture of released hemoglobin duringintravascular hemolysis
Our recent data revealed that Hp isolatedfrom serum of HCC patients carries up to six fucoses per glycan
In the present study, we have used neuraminidaseassisted LC–MS/MS to confirm that all glycosites of Hp carrycomplex glycans with variable proportion of bi-, tri-, and tetra-antennarystructures and variable degree of fucosylation (Table 1)
Tri-antennary structures on peptide V(218) –K(233)are as abundant as bi-antennary structures
To further refine structureassignments, we have used MSn analysisof detached permethylated N-glycans
Our previous study used glycosidase-assistedLC–MS/MS to show that hyper-fucosylated glycoforms of Hp containmostly Lewis-X- and Lewis-Y-type glycoforms
Detailed MSn structural analysis of tetra-antennary doubly fucosylatedglycan with monosaccharide com position (Hex)4 (HexNAc)4 (Deoxyhexose)2(NeuAc)2 \+ (Man)3(GlcNAc)2 supports thepresence of Lewis X, H2, and Lewis Y glycoforms
Dissociation (MS2 ) of this doubly fucosylated permethylated glycan at m/z 1366 (3+) gives Lewis type fragmentsat m/z 834 and 660
Both fragmentswere further dissociated ( MS3 ) and product ion at m/z 646 (derived from 834 fragment ) wasfurther collided (MS4)
MSn analysis shows at least threepossible isomers at this com position, with both core and antennalfucosylation
Figure 2 shows selected MSn spectrafor the reduced and permethylated tetra-antennary, disialylated, anddoubly fucosylated com position (m/z 1366 (3+))
Figure 2A shows the MS2 spectrumof this com position, with insets displaying zoomed in views of theterminal (B2) and internal (B3/Y) lactosamine fragments as well asthe Y1 fragment (m/z 490 ) indicativeof the fucosylated reducing-end GlcNAc; the second inset shows theterminal doubly fucosylated lactosamine (m/z 834) and the sialylated lactosamine (m/z 847 ) fragments
The m/z 490 fragment indicates traces of core fucosylated glycans
The MS2 spectrum also clearly indicates two different antennal fucosylationfragments : the terminal (B-type) monofucosylated fragment at m/z 660 and the terminal ( B-type ) difucosylatedfragment at m/z 834
Because thereare several structural isomeric possibilities for these fragments ,these ions were subjected to further MSn interrogation
The MS3 fragmentationof the m/z 660 ion (Figure 2B) is indicative of a mixture of Lewis X and H2epitopes (Figure 2)
These isomeric glycansdiffer in fucose position and linkage and are particularly difficultto separate by LC–MS analysis; MSn fragmentation is a complementarymethod that resolves the linkage efficiently
The specific diagnosticfragments are the m/z 259, a C-typeterminal hexose that can arise from a Lewis X structure but not anH2 structure, m/z 329, a 3,5A cross-ring cleavage fragment that can only arise from Lewis X,not H2 or Lewis A, and the m/z 503 ,another 3,5A cross-ring cleavage fragment which can onlyarise from H2, not H1 or Lewis A or X. Detailed structures with fragmentidentifications are in the Supporting Information
The low-intensity fragment at m/z 834 (terminal difucosylated lactosamine) in the MS/MS spectrum isconsistent with the possible Lewis Y epitope
Disassembly of thision through MS3 , m/z 834 (Figure 2C) and MS4, m/z 834 → 646 (Figure 2D) are consistentwith the Lewis Y epitope
The MS3 andMS4 spectra both contain the m/z 503 fragment , a 3,5A cross-ring cleavage ion, of thesame structure as that found in H2, which distinguishes this fromLewis B. Because this fragment is ofthe same structure as that from H2, the fragmentation pathway, throughthe m/z 834 ion, is of great importancein assigning this structure
We were not able to detect any core-fucosylatedglycoforms in Hpby LC–MS/MS
The low abundant core-fucosylated glycoform detectedby MSn (see above) is either a minor component below the detectionlimit of our LC–MS/MS analysis or derives from a trace-contaminating protein
We cannot exclude that low percentage of core fucose existsespecially on the doubly fucosylated site , but it is clear that core-fucosylatedglycoforms represent at most a very minor component of the Hp glycoforms
Hemopexin : Hpx is a heme-binding 60 kDa plasma glycoprotein containingfive N-glycosylation sites and one N-terminal O-glycosylation site
Its major biological role is defense against hemoglobin-mediated oxidativedamage during intravascular hemolysis
We have analyzed microheterogeneity of three tryptic N-glycopeptidesof Hpx
Each glycopeptide is occupied by complex glycans, primarilybi- and tri-antennary glycoforms with up to two fucoses
Bi-antennaryglycans dominate, and their fucosylated counterparts represent a minor(<10%) contribution (Table 1)
One tetra-antennaryglycoform with three fucoses was observed at SWPAVGNCSSALRpeptide , but multiply fucosylated and highly branched structures arerare on Hpx , contrary to Hp
Although the ion intensities of mostsingly fucosylated bi-antennary glycopeptides were not sufficientfor fragmentation , the measurable CID spectra contain low-intensitypeptide-HexNAc-Fuc fragment consistent with core fucosylation
Thepresence of oxonium ions at m/z 512.2indicates the presence of outer-arm fucose
The intensity of fragments ,which could distinguish fucose linkage in doubly and triply fucosylatedglycoforms, was under the detection limit of our instrument
Fragmentationspectra of SWPAVGNCSSALR glycopeptide bearing A3G3F1and A3G3F2 glycans contain both oxonium ion m/z 512.2 consistent with Lewis X type linkage
The presenceof oxonium ion at m/z 658.2 in fragmentationspectrum of the A3G3F2 glycopeptide could suggest the presence ofLewis-Y-type structure, but rearrangement of fucose at outer armsis a likely event; independent methodswould have to confirm such linkage assignment
We were not able toconfirm Lewis Y linkage by MSn analysis and conclude that rearrangementof the Lewis X structures is more likely in this case
This fucoserearrangement, however, was not described to happen between core andouter arms in tri- and tetra-antennary glycans, and we can concludethat core fucosylation is minor and below the limit of our detectionin the tri-antennary structures
Theseassignments are quite consistent with analysis of N-glycans detachedfrom Hpx in a study of cirrhotic and HCC patients
Debruyne et al. observed intense bi-antennary glycan withsmall contribution of fucosylation and minor tri- and tetra-antennaryglycoforms compared with the dominant bi-antennary structure
Theauthors found that outer-arm fucosylated tri- and tetra-antennaryglycans are increased in HCC compared with bi-antennary glycan withoutfucose and suggest that their ratio can be used as a diagnostic testfor detection of HCC
MALDI-Tof spectrum of glycans releasedfrom isolated Hpx is dominatedby bi-antennary structures (Supplemental Figure1 in the Supporting Information)
Disassembly of a bi-antennary,monosialylated, monofucosylated com position shows a mixture of corefucosylation, Lewis X, and sialylated Lewis X-containing isomers (Supplemental Figure 4 in the Supporting Information)
Kininogen-1 : Kng-1 , a plasma glycoprotein of 644 amino acids,containsa heavy chain with four N-glycosylation sites
Serine proteases cleave from Kng-1 bradykinin , a nine amino acidpeptide that regulates many biological processes including inflammation,angiogenesis, and cell migration
Wehave identified bi- and tri-antennary glycans and their fucosylatedforms at three of four N-glycosylation sites of Kng-1
The glycopeptideYNSQNQSNNQFVLYR was observed only with singleglycoform A2G2
However, the intensity of this peptide is very low,and we cannot exclude the presence of other glycoforms below the detectionlimit of our method
Two glycopeptides carry tri-antennary glycoformswith up to two fucoses per glycan; moreover, glycopeptide HGIQYFNNNTQHSSLFMLNEVKRcarries tetra-antennary glycans with up to five fucoses (Figure 3A)
This is similar to the multiply fucosylatedglycoforms of Hp and in contrast with the limited fucosylation ofglycoforms associated with Hpx and CFH
Figure 3B shows a representative CID fragmentation spectrum of triply fucosylatedtetra-antennary glycoform of HGIQYFNNNTQHSSLFMLNEVKRpeptide with glycan fragments consistent with Lewis X structure at m/z 512.2
There is no evidence of corefucosylation in Kng-1 glycopeptides based on their CID fragmentation
Unfortunately, we were not able to complete MSn structural analysisof Kng-1 ’s glycans because the protein was not available insufficient amount and purity
Complement factor H : CFH is a 140 kDa (without carbohydratemoiety)plasma glycoprotein that regulates enzymatic activity of complementC3
CFH has nine N-glycosylation sequonsand is primarily secreted to plasma by the liver even though a membranebound form exists as well
We have detectedseven glycopeptides in the tryptic digest of CFH which cover eightsequons (one peptide is doubly glycosylated)
All peptides carry alimited variety of glycoforms of the complex type; high mannose orhybrid glycoforms were not detected
We have observed dominant bi-antennaryglycoforms on all sequons except peptide L(528)-R(567), which carriesmore intense tri-antennary glycoforms
The bi-antennary glycoformsare singly fucosylated, but the intensity of the fucosylated glycoformsis somewhat higher (up to 20%) than for Hpx (<10%)
Similar to Hpx , the singly fucosylated tri-antennary glycoforms tend to havehigher intensity than their nonfucosylated counterparts
We have detectedthe minimal amount of doubly fucosylated glycoforms; tetra-antennaryglycoforms and glycoforms with more than two fucoses were not detected(Table 2)
Structural analysis of detachedpermethylated glycans uncovered the presence of core-fucosylated glycansrepresented by Fuc-GlcNAc fragment at m/z 490 (Supplemental Figure 3 in the SupportingInformation)
The intensity of this fragment is roughly similarto the fragment at m/z 660 correspondingto LeX epitope
This is in contrast with other analyzed singly fucosylatedglycoforms from Hpx and Hp, where the 660 fragment was dominant, andthe 490 fragment was small but detectable
These findings demonstratethat the process of glycan branching and fucosylation is site-specificand differs significantly between different glycoproteins isolatedfrom the same patient

Output (sent_index, trigger, protein, sugar, site):

1. glycopeptide, , -, -, glycopeptide
11. glycoprotein, , Haptoglobin, -, -
11. glycoprotein, , glycoprotein, -, -
14. glycosites, , -, -, glycosites
22. disialylated, , -, -, position
22. fucosylated, , -, -, position
23. fucosylated, , -, the fucosylated reducing-end GlcNAc, -
23. sialylated, , 847, -, fragments
25. monofucosylated, , fucosylationfragments, -, fragment
34. core-fucosylated, , MSn, -, -
35. fucosylated, , -, -, site
36. N-glycosylation, , -, -, sites
36. O-glycosylation, , -, -, site
36. glycoprotein, , Hemopexin, -, -
36. glycoprotein, , Hpx, -, -
36. glycoprotein, , glycoprotein, -, -
38. microheterogeneity, , N-glycopeptidesof Hpx, -, -
39. glycopeptide, , -, -, glycopeptide
39. occupied, , -, -, glycopeptide
42. fucosylated, , -, -, glycopeptides
42. glycopeptides, , -, -, glycopeptides
45. glycopeptide, , -, -, glycopeptide
46. glycopeptide, , -, -, glycopeptide
54. N-glycosylation, , -, -, sites
54. glycoprotein, , Kng-1, -, -
54. glycoprotein, , glycoprotein, -, -
56. N-glycosylation, , Kng-1, -, sites
59. glycopeptide, , -, -, glycopeptide
59. glycopeptides, , -, -, glycopeptides
61. glycoform, , -, -, HGIQYFNNNTQHSSLFMLNEVKRpeptide
62. glycopeptides, , -, -, glycopeptides
64. glycoprotein, , CFH, -, -
64. glycoprotein, , glycoprotein, -, -
66. glycopeptides, , -, -, glycopeptides
66. glycosylated, , -, -, peptide
67. glycoforms, , -, high mannose orhybrid glycoforms, -
7. glycoprotein, , glycoprotein, -, -
70. fucosylated, , Hpx, -, -
75. glycoproteins, , glycoproteins, -, -
8. analyzeglycopeptides, , Kng-1, -, analyzeglycopeptides
9. glycosites, , -, -, glycosites
9. microheterogeneousglycopeptides, , -, -, microheterogeneousglycopeptides

Output(Part-Of) (sent_index, protein, site):

11. glycoprotein, fourN-glycosylationsites
20. 834, fragment
23. Y1, fragment
24. 490, fragment
55. Kng-1 bradykinin, acidpeptide
56. Kng-1, sites
62. Kng-1, glycopeptides
73. fragment, fragment
74. 490, fragment
8. Kng-1, analyzeglycopeptides
9. protein, glycosites

*Output_Site_Fusion* (sent_index, protein, sugar, site):