Section : Purification of ITIH4 fromCell Media and Human Serum
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Recombinant ITIH4 was isolatedfrom the cell media of HEK293 cells overexpressing ITIH4-cMyc-DDKprotein by ultrafiltration followed by RP-1S chromatography
Afterthese steps, ITIH4 exceeded 90% purity based on SDS-PAGE with Coommassieblue staining (data not shown)
Proteomic analysis of a tryptic digestconfirmed the identity of ITIH4 (71% sequence coverage) with minimalprotein impurities
To purify ITIH4 from serum, we devised a three-steppurification process to minimize biased selection of specific glycoformsby the chromatographic steps (Figure 1A)
Ammoniumsulfate precipitation resulted in a 5-fold enrichment of ITIH4 witha yield of 84% (Table 1)
Further enrichmentwas achieved by Cibacron Blue chromatography (3-fold enrichment) followedby reversed-phase chromatography on a monolithic protein purificationcolumn (10-fold enrichment)
The observed mass shift of recombinantand serum ITIH4 on SDS-PAGE after treatment with PNGaseF confirmedthat ITIH4 was heavily N-glycosylated (Figure 1B)
Section : N-Glycan Site Occupancyof Serum and Cell-Derived ITIH4
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To confirm that the fourcanonical N-glycosylation motifs in ITIH4 were glycosylated, we treated ITIH4 glycopeptides (enriched using HILIC chromatography) with PNGaseFin the presence of H218O
To avoid 18O incorporation into the peptide C-terminus during trypsin digestion,we inactivated proteases by heating and addition of protease inhibitorsprior to PNGaseF/18O labeling
18O-Labeled b- and y-ions in the peptide MS/MS spectraenabled residue-specific resolution of the N-glycosylation sites
Peptides containing the four consensus sequence N-glycosylation siteswere detected with the expected +3 Da mass shift
The MS/MS spectraof 18O-labled peptides confirm the expected glycosylationof the N81, N207, N517, and N577 sites within the NXS/T motifs
Wealso detected a fifth peptide , NVVFVIDK, with a +3 Da shift,in both recombinant and serum ITIH4
The b- and y-ions in the MS/MS spectrum of the (N > D/18O)VVFVIDK peptide definitively confirm that the +3 Da shift is locatedat N274
Figure 1C shows a schematic of theconfirmed ITIH4 glycosylation sites
To provide a quantitativeestimate of occupancy at each ITIH4 N-glycosylation site , we appliedthis same PNGaseF/H218O isotopic labeling strategyto ITIH4 digests, as previously described
Importantly, while the N-glycosylation site confirmation experimentsabove were performed on enriched glycopeptide fractions, these occupancyexperiments were performed on unprocessed (non-HILIC enriched) ITIH4digests
This enabled us to estimate the glycosylation site occupancyby comparing the intensity of an 18O-labeled peptide (whichwas formerly glycosylated) with the unlabeled (and therefore nonglycosylated) peptide containing the same N-glycosylation site
Quantification ofthe non-glycosylated and corresponding deglycosylated peptides inextracted ion chromatograms (XIC) of ITIH4 digests treated with PNGaseF/H218O shows that at least three of the four consensusglycosylation sites ( N207, N517, and N577 ) are highly occupied (>90%)in serum-derived ITIH4 (Table 2)
We observetwo labeled (+3 Da) semispecific peptides containing site N81 , AFIT(N> D/18O)F ( residues77–82 ) and KAFIT(N > D/18O)F ( residues 76–82), in ITIH4 trypsin- GluC digeststreated with PNGaseF/H218O
We were unable toconfidently quantify the occupancy of site N81 in serum ITIH4 becausethe non-glycosylated peptides containing site N81 are below the limitof detection in serum-derived ITIH4 digests
In recombinant ITIH4 , site N81 is partially occupied (>80%) and sites N207, N517 , andN577 are highly occupied (>90%)
Occupancy of the noncanonicalNVV motif is low (<1%) in both serum and recombinant ITIH4
Theseobservations are also confirmed by glycopeptide MS/MS data for bothrecombinant and serum-derived ITIH4 (Figure 2)
These results demonstrate that N-glycosylationsite occupancy of the five ITIH4 N-glycosylationsites is similar in serum and recombinant ITIH4
Section : N-Glycan Microheterogeneity in Recombinant ITIH4
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To achievesite-specific characterization of N- and O-glycan microheterogeneity,we performed serial proteolysis ( GluC-trypsin and trypsin-chymotrypsin )of ITIH4 , enriched glycopeptides using HILIC chromatography, and analyzedglycopeptides via LC– ESI-MS/MS before and after treatment withneuraminidase and fucosidase
All observed glycoforms are summarizedin Table 3
In recombinant ITIH4 , biantennarysialylated glycans were the dominant glycoforms at N81 ; triantennary,fucosylated, and high-mannose forms were also observed
At sites N207,N517N207,N517, and N577 , complex fucosylated, asialo-glycans were the dominantglycoforms, though some sialylated glycoforms were also observed
At N207 , all detected glycoforms were fucosylated, and several glycopeptideswith bifucosylated N-linked glycans were detected
We observed thegreatest microheterogeneity at N517 , with nonfucosylated, monofucosylated,and bifucosylated bi-, tri-, and tetra-antennary N-linked glycans
At N577 both sialylated and asialo-glycoforms were observed, as wellas singly fucosylated N-linked glycans
Analysis of detached, permethylatedN-linked glycans from recombinant ITIH4 corroborates the glycan com positions matched to glycopeptides from CID MS/MS
The detached glycan dataalso confirmed the abundance of fucosylated glycoforms—fucosylatedN-linked glycans accounted for 89% of the relative intensity of permethylatedglycans analyzed via MALDI-TOF MS (SupplementaryFigure 1, Supporting Information)
Double-fucosylated N-linkedglycans contributed 3% of the N-glycan relative intensity
Fully sialylatedN-linked glycans accounted for only 13% of the signal, confirmingglycopeptide results that also demonstrated that fully sialylatedglycans are a minor component of recombinant ITIH4 N-glycoforms
Notably,we observed the M5 N-linked glycan in MALDI-TOF spectra, representing0.1% of the relative intensity, consistent with our previous observationthat high-mannoseglycopeptides are very low in abundance
We usedoptimized methods for analysis of recombinant ITIH4 to study site-specificglycoforms in serum-derived ITIH4
Section : N-Glycan Microheterogeneity in Serum-Derived ITIH4
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Analysis of serum-derived ITIH4 glycopeptides showedthat all canonical glycosylation sites in serum-derived ITIH4 containedcomplex sialylated N-linked glycans (Table 3)
Analysis of detached, permethylated N-linked glycans from serum-derived ITIH4 was consistent with glycopeptide results (data not shown)
Glycosylationsites N81 and N207 , located closest to the N-terminus of the protein,carried primarily bi- and triantennary sialylated glycans; a higherdegree of microheterogeneity was present at the N517 and N577 siteswith additional fucosylated and tetra-antennary sialylated glycansobserved in addition to the dominant bi- and triantennary sialylatedforms
We detected the highest number of glycoforms, and highly branchedtetra-antennary glycans, at the N517 site
A CID fragmentation spectrumof [LPTQNITFQTE + A2G2S2 + 4H]4+ (m/z 874.9) glycopeptide is shown in Figure 2A
Oxonium ions, including [HexNAc + H]1+ at m/z 204, [NeuAc – H2O + H]1+ at m/z 274, [Neu5Ac + H]1+ at m/z 292, [HexNAc-Hex + H]1+ at m/z 366 and [HexNAc-Hex-Neu5Ac + H]1+ at m/z 657 in the spectrum are diagnosticof glycopeptide fragmentation and offer clues regarding glycan topology;intact peptide peaks including [LPTQNITFQTE + H]1+ at m/z 1291.65, and [LPTQNITFQTE+ HexNAc + H]1+ at m/z 1494.73 enable the assignment of peptide identity and the determinationof the N-linked glycan com position ; and peptide b and y ions (y2: m/z 249.11, 1+; b4: m/z 440.25, 1+; b5: m/z 554.29, 1+; b9: m/z 1043.54, 1+; and b10: m/z 1144.59,1+) corroborate the identity of the peptide
Consistent with our observation that glycosylationwas present at N274 based on PNGaseF/18O labeling (N >D/18O), we also detected four high-mannose glycoforms ofthe NVVFVIDK peptide , including M5, M6,M7, and M8 glycoforms in recombinant ITIH4 , and M5 and M6 glycoformsin serum-derived ITIH4
The CID spectrum of serum-derived glycopeptideNVVFVIDK + M6 (Figure 2B) is representativeof CID MS/MS spectra from both serum and recombinant ITIH4 and offersdefinitive evidence of glycosylation at this noncanonical site
Thespectrum contains oxonium ions including [HexNAc + H]1+ at m/z 204, [Hex-Hex + H]1+ at m/z 325, [HexNAc-Hex\+ H]1+ at m/z 366, and[HexNAc-Hex-Hex + H]1+ at m/z 528 consistent with a high-mannose glycan and a strong peptide b- and y-ion series (b2-b5 and y2-y6) leading to a confidentassignment
In addition, a series of glycopeptide Y ions including[NVVFVIDK + HexNAc2Hex1 + 2H]2+ at m/z 751.33, [NVVFVIDK+ HexNAc2Hex2 + 2H]2+ at m/z 832.34, [NVVFVIDK + HexNAc2Hex3 + 2H]2+ at m/z 913.41, [NVVFVIDK + HexNAc2Hex4 + 2H]2+at m/z 994.47, and [NVVFVIDK + HexNAc2Hex5 + 2H]2+ at m/z 1075.43,consisting of the intact peptide and partially fragmented glycan,and B ions including [HexNAc1Hex3 + H]1+ at m/z 690.19, [HexNAc1Hex6 + H]1+ at m/z 1176.35 consisting of the fragmented glycan, are alsopresent in the spectrum
ITIH4 glycopeptides from the recombinant ITIH4 demonstrate a greater variety of the high-mannose glycoforms(M5-M8) (Table 3), but the glycoforms in serumare limited to high-mannose forms as well
Section : Core- and Outer-Arm Fucosylationof ITIH4 N-Linked Glycans
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On the basis of glycopeptide fragmentationdata , evidence suggests that most fucosylated recombinant ITIH4 glycoformsare core fucosylated while most fucosylated serum ITIH4 glycoformsare outer-arm fucosylated
The CID spectra of fucosylated N-glycopeptidesfrom recombinant ITIH4 contained [HexNAc-Fuc + H]1+ peaksat m/z 350, and peptide-specificpeaks equivalent to the mass of the peptide with HexNAc-Fuc , whichsuggests that these glycopeptides contain core fucosylated glycans
However, we also detected [HexNAc1Hex1Fuc1 + H]1+ ions at m/z 512 in a minority of spectra from recombinant ITIH4 , suggestingminor amounts of outer-arm fucosylation may also be present
Becauserearrangement of fucose during fragmentation from core to outer armis unlikely, a likely explanation isthat the glycopeptides of recombinant ITIH4 represent a mixture ofouter-arm and core fucosylated peptides
In serum-derived ITIH4 , weonly observed fucosylated glycopeptide ions at sites N517 and N577
However, we observed both [HexNAc1Hex1Fuc1 + H]1+ at m/z 512 (outer-arm), [HexNAc-Fuc + H]1+ at m/z 350 (core), and [HexNAc1Hex1Fuc1Neu5Ac1 + H]1+ at m/z 803 in MS/MS spectra consistentwith the presence of N-glycans with sialylated outer-arm linked fucose
This suggests that serum ITIH4 , like recombinant ITIH4 , contains bothcore- and outer arm-fucosylated N-linked glycans
In vertebrates,fucose is linked to the N-glycan core via an α1–6 linkage,whereas outer-arm fucoses are linked via α1–3 linkage
Therefore, treatment with α( 1–3,4 ) fucosidase shouldremove outer-arm fucoseresidues but should not cleave core fucose
In recombinant ITIH4 most fucosylated glycopeptides were resistantto α( 1–3,4 ) fucosidase , indicating that recombinant ITIH4fucosylated glycoforms are core fucosylated
We observed the nearcomplete disappearance of three doubly fucosylated glycopeptides ALTTWQNK+ FA2BF1G1, LPTQNITFQTE + FA2F1G2, and LPTQNITFQTE+ FA2BF1G1 in recombinant ITIH4 digests after α( 1–3,4 ) fucosidase treatment
This presumably occurred due to the conversionof these forms to singly fucosylated forms after removal of α1–3linked fucose on the outer arms of the glycans
These results confirmedthat recombinant ITIH4 glycopeptides were highly core-fucosylated,but some also contained outer-arm fucosylation
The ratio of the fucosylatedto nonfucosylated forms of recombinant ITIH4 glycopeptides LPTQNITFQTE+ A2G2 and LPTQNITFQTE + A3G3 remained virtually unchangedafter treatment with α( 1–3,4 ) fucosidase in recombinant ITIH4 (Figure 4E–H); the peak area ofthe unglycosylated LPTQNITFQTE + A2G2 glycopeptide is1.4% of the LPTQNITFQTE + FA2G2 precursor in untreatedglycopeptides , versus 1.7% after treatment
A similar pattern wasobserved for LPTQNITFQTE + A3G3 and LPTQNITFQTE+ FA3G3, with the unfucosylated form representing <2% of the fucosylatedform before and after fucosidase treatment, indicating that recombinant ITIH4 is predominantly core-fucosylated
In serum-derived ITIH4 ,fucosylated biantennary glycoforms are predominantly core-fucosylatedwhile triantennary fucosylated glycoforms contain higher levels ofouter-arm fucosylation
The ratio of the fucosylated glycopeptideLPTQNITFQTE + FA2G2 to the unfucosylated LPTQNITFQTE+ A2G2 glycopeptide was 4.7% prior to treatment with α( 1–3,4 ) fucosidase (Figure 4A)
We observed very littlechange in the ratio of the intensities of serum-derived ITIH4 glycopeptidesLPTQNITFQTE + FA2G2 and LPTQNITFQTE + A2G2before and after treatment with α( 1–3,4 ) fucosidase (Figure 4A,B), indicating that this glycopeptide containscore-linked fucose
In contrast, we observed that the precursor intensityof LPTQNITFQTE + FA3G3 was 33% of the intensity of theunfucosylated form LPTQNITFQTE + A3G3 prior to fucosidasetreatment, whereas after treatment this ratio dropped to 4.5% (Figure 4C,D)
Therefore, the fucosylated triantennary glycopeptidecontaining site N517 is a mixture of outer-arm fucosylated and core-fucosylatedforms
On the basis of the α( 1–3,4 ) fucosidase treatmentthe predominant glycoform is outer-arm fucosylated and a small amount(4.5%) of the core-fucosylated glycoform is also present
In conclusion,core fucosylation dominates the N517 biantennary glycan,while outer arm fucosylation represents a majority of the triantennary fucosylated glycoforms in serum ITIH4 , andat site N577 the majority of fucosylated glycoforms contain outer-armfucosylated glycans
Section : O-Glycopeptide Microheterogeneity in Recombinant ITIH4
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We also characterized several O-glycopeptides in recombinantand serum-derived ITIH4
Recombinant ITIH4 is glycosylated on overlappingpeptides IPKPEASFSPR ( residues 634–644) and ASFSPR( residues 639–644) containing potentially glycosylated residuesS640 and S642
We identified two major glycoforms of this peptide ,including HexNAc1Hex1NeuAc1 and HexNAc1HexNAc1NeuAc2, and several minor glycoforms (Table 3)
To complement these analyses, we selectively detached O-glycans fromrecombinant ITIH4 via reductive beta-elimination and then performedMS and MS/MS analyses on detached, permethylated O-glycans (Supplementary Figure 1B, Supporting Information)
We detected simple O-glycan structures, including HexNAc1Hex1NeuAc1 and HexNAc1HexNAc1NeuAc2, equivalent to theglycan com positions on IPKPEASFSPR and ASFSPR
Therefore,we suggest that it is likely that only one of the potential O-glycosylationsites on this peptide (either S640 or S642) is glycosylated
We alsoidentified a second glycopeptide , GPDVLTATVSGK, spanningresidues 501–512, with multiple glycoforms (Table 3), including HexNAc1Hex1NeuAc2 and HexNAc2HexNAc2NeuAc2.
Because we detect only simple O-glycans in our detachedglycan analysis, and we detect glycopeptides with higher com positions on glycopeptide GPDVLTATVSGK, we suggest that at leasttwo of the potentially O-glycosylated residues on this peptide (T506,T508, S510) are glycosylated with simple O-glycans
We also detectedtwo fucosylated O-glycopeptides on GPDVLTATVSGK from recombinant ITIH4
We did not detect fucosylated O-glycans in our detached glycananalyses, suggesting that fucosylated O-glycans were below the limitof detection
The recombinant ITIH4 protein sequence differsfrom the canonical sequence in UniProt (Q14624) at residues 85 (Ito N) and 698 (P to T)
We detect multiple glycoforms of peptide LAILPASATPATSNPDPAVSR ( residues 690–710)
NonsialylatedO-glycopeptides including HexNAc1, HexNAc2,HexNAc1Hex1, HexNAc2Hex2, HexNAc3Hex4 and HexNAc4Hex4 account for over half of the glycoforms (basedon relative intensity) of this peptide ; we also detect com positions consistent with simple sialylated O-glycans including HexNAc1Hex1NeuAc2, HexNAc3Hex3NeuAc3, and HexNAc4Hex4NeuAc4 (Table 3A)
In addition, we detect a third (partially overlapping) glycopeptide ,PAVSRVMNMK + HexNAc1HexNAc1NeuAc2 in trypsin-chymotrypsin digests of recombinant ITIH4
This peptide contains a single potential glycosylation siteat S709
We have identified multiple O-glycoforms on three differentpeptides in trypsin- GluC and trypsin-chymotrypsin digests of recombinant ITIH4
Section : O-Glycopeptide Microheterogeneity in Serum-Derived ITIH4
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We also characterized O-glycopeptides in serum ITIH4 by CID MS/MSand detached O-glycan analyses
Glycopeptide IPKPEASFSPR+ HexNAc1Hex1NeuAc1 ,and the doubly sialylated [IPKPEASFSPR + HexNAc1Hex1NeuAc2 + 3H]3+ format m/z 726.01 with one missed GluCcleavage (at glutamic acid ) were observed in both serum and recombinant ITIH4
An alternately cleaved glycopeptide , ASFSPR, consistent withcleavage by GluC at the glutamic acid residue , is observed with thesame glycan com positions, including [ASFSPR + HexNAc1NeuAc1 + 2H]2+m/z 579.78,[ASFSPR + HexNAc2Hex2 + 2H]2+ at m/z 653.66, [ASFSPR + HexNAc1Hex1NeuAc1 + 2H]2+ at m/z 660.79, and [ASFSPR + HexNAc1Hex1NeuAc2 + 2H]2+ at m/z 806.36, as summarized inTable 3B
A CID fragmentation spectrum of[ASFSPR + HexNAc1Hex1NeuAc2 + 2H]2+ (m/z 806.36)in Figure 3B shows multiple glycopeptide Yions with the intact peptide and a partially fragmented glycan attachedto the peptide backbone
There are two serine residues in this peptide (S640, S642), each representing a potential site of glycan attachment
On the basis of CID fragmentation alone, we are unable to determineif glycosylation is restricted to a single site
However, the com positions of serum-derived ITIH4 detached O-glycans (SupplementaryFigure 1C, Supporting Information) suggest that the peptideis glycosylated at a single site
In ITIH4 purified from pooledhuman serum we detect glycoforms of both LAILPASAPPATSNPDPAVSR and LAILPASATPATSNPDPAVSR, indicating that both the canonical ITIH4 sequence and the P698 to T variant are present in the sample
The glycan com positions observed on LAILPASAPPATSNPDPAVSR in serum-derived ITIH4 include HexNAc2Hex2NeuAc2, HexNAc2Hex2NeuAc3, HexNAc3Hex3NeuAc3, HexNAc3Hex3NeuAc4, and HexNAc4Hex4NeuAc4 and are similar to those observed on the T698 variant peptide (Table 3)
Despite thedifference in sequence , the most common glycoforms (HexNAc2Hex2NeuAc2 and HexNAc3Hex3NeuAc3) associated with bothpeptides have similar com positions and relative intensities in serum ITIH4 (Table 3)
Potential O-glycosylatedresidues include S696, T701, S702, and S709
We detected mostly simpleO-glycan structures in detached O-glycan analyses of serum-derived ITIH4 (Supplementary Figure 1, Supporting Information)
We also detected more complex O-glycans in detached O-glycan analyses,but these represented minor components of the mixture
The most likelyexplanation for these observations is that the observed glycan com positions, including HexNAc3Hex3NeuAc3, and HexNAc3Hex3NeuAc4, represent the sum of multiple smaller O-glycans divided among threeglycosylated residues in the peptide
In the glycopeptide CID MS/MSspectrum of [LAILPASAPPATSNPDPAVSR + HexNAc3Hex3NeuAc3 + 4H]4+ (m/z 1004.2) from serum-derived ITIH4 shown in Figure 3A the y5 ion (m/z 529.31, 1+) as well as the y5 ion with HexNAc(m/z 732. 39, 1 +) andHexNAc-Hex (m/z 894.44, 1+) are clearly present
This strongly supports that one of the sitesof glycosylation is S709, which is further corroborated by evidenceof glycosylation on S709 in recombinant ITIH4
We also observe theseions (y5 at m/z 529.31, 1+; y5 \+HexNAc at m/z 732. 39, 1 +; and y5 + HexNAc-Hex at m/z 894.44, 1+) in the P698 to T variantglycopeptides in serum and recombinant ITIH4 , indicating that in allcases S709 is glycosylated
To further explore the sites of O-glycosylation in serum-derived ITIH4 , we used targeted electron-transfer dissociation (ETD) on ITIH4glycopeptides
As opposed to most of our CID analyses, ETD of glycopeptidesresults in fragmentation of the peptide backbone rather than of glycosidiclinkages
Peptide c- and z-ionsresulting from ETD fragmentation of glycopeptides retain unfragmentedcarbohydrate moieties, which can be used to determine the site(s)of glycosylation
We labeled the serum-derived ITIH4 glycopeptideswith mTRAQ reagents to boost the charge, treated them with a broad-specificityneuraminidase to remove sialic acids, and then subjected them to ETD(Supplementary Figure 4, Supporting Information)
On the basis of the MS2 spectrum of glycopeptide IPKPEASFSPR,S640 is O-glycosylated, while S642 is not
This is supported by thepresence of c7, c8, and c9 and z5, z6, and z7 ionswith HexNAc-Hex in the fragmentation spectrum, as well as the absenceof this modification on z3, z4, c5, and c6 ions
ETD fragmentation of the desialylated, mTRAQ-labeledLAILPASAPPATSNPDPAVSR + HexNAc2Hex2peptide from serum ITIH4 indicates that theselected glycopeptide is glycosylated at S696 and T701, based on c- and z-ions in the MS2 spectrum (Supplementary Figure 4, Supporting Information)
We were unable to obtain information on the desialylated LAILPASAPPATSNPDPAVSR+ HexNAc3Hex3glycopeptide , so we arenot able to corroborate the CID results that also indicate that S709is O-glycosylated
On the basis of the available information, themost likely explanation is that LAILPASAPPATSNPDPAVSRis O-glycosylated at S696, T701 and S709
We detect similar glycancom positions associated with peptides LAILPASATPATSNPDPAVSR and LAILPASAPPATSNPDPAVSR in serum ITIH4 , suggesting thatthe sequence variant is not glycosylated differently
Section : Purification of glycoprotein ITIH4 from human serum
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(A) Purificationschema of ITIH4 from serum
(B) Western blot (anti- ITIH4 , SC-21987)of ITIH4 overexpressed and purified from HEK293 cells ( Cell ITIH4 )and from serum (serum ITIH4 )
Lane 1: Cell-derived ITIH4 ; lane 2:Cell ITIH4 treated with PNGaseF; lane 3: protein ladder with proteinmolecular weights indicated to the left of the blot; lane 4: serum ITIH4 ; lane 5: serum ITIH4 treated with PNGaseF
Section : ITIH4 is a secreted glycoprotein that is up-regulated by IL-6 aspart of the acute phase response to turpentine-induced inflammationin rats and in response to infection in humans
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Glycosylation of ITIH4 is of considerableinterest due to the biomarker potential of its proteolytic productsand glycoforms in several forms of cancer, the involvement of ITIH4in liver development and stabilization of the ECM , and observed changesin ITIH4 expression and glycosylation in ovarian and other cancers
Glycosylation can mediate protein interactions, influence protein stability, and impact proteinfunction
Our results confirm previous reports that ITIH4 is heavilyglycosylated; it is therefore plausible that glycosylation modifies ITIH4 structure and function
While microheterogeneity of glycoformsdiffers between ITIH4 protein expressed in HEK293 cells and proteinisolated from serum, occupancy of N-glycosylation sites does not differ
However, utilization of O-glycosylation sites differs between thecell line and serum
We have determined occupancy of each glycosylationsite quantitatively by treatment of the N-glycopeptides by PNGaseFin the presence of H218O
Our results show thatat least three of the four canonical N-glycosylation sites ( N207,N517N207,N517, and N577 ) are highly occupied >90% occupancy) in serum-derived ITIH4 , and a similarly high degree of glycosylation was observed onthe recombinant ITIH4
We also confirm that site N81 is glycosylated
N-Glycosylation site occupancy is thought to depend on the primarysequence of the protein because the transfer of precursor glycansfrom dolichol donors to asparagine residues occurs prior to proteinfolding
The observed similarity in occupancy betweenrecombinant and serum-derived ITIH4 is consistent with this hypothesisand suggests that cell culture might be a representative model fordetermination of site occupancy of protein glycoforms
However, variationsin cell culture conditions, including changes in temperature, pH,and the availability of metal ions, reportedly affect N-glycosylationsite occupancy
It is interesting tonote that the noncanonical glycosylation site at position N274 haslow occupancy in both recombinant and serum-derived ITIH4
Section : Site-SpecificMicroheterogeneity of ITIH4 N-Glycoforms
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Contrary to siteoccupancy, the microheterogeneity of N-glycoforms differs substantiallybetween recombinant and serum-derived ITIH4
On the basis of CID MS/MSof untreated and exoglycosidase-treated glycopeptides and MALDI-TOFanalysis of detached, permethylated N-glycans, we conclude that complex,core fucosylated, asialo-glycoforms are dominant in recombinant ITIH4
Fully sialylated forms represent a minor component of all N-glycoformsat allsites except N81
This is also the only canonical glycosylationsite with detectable high mannose glycans
Core-fucosylated glycansat N81 represent a minor component of the total microheterogeneity
This is in contrast to the glycoforms at the three other canonicalglycosylation sites ( N207, N517, and N577 ) of recombinant ITIH4 , wherecore-fucosylated forms dominate
We also observed more highly branchedN-linked glycans, including tetra-antennary glycans, at N517 and N577
Of the four canonical sites , we observed the highest microheterogeneityof glycoforms at N517
This may be due in part to the efficiency ofour analytical workflow for this glycosylation site ; for example,the LPTQNITFQTE peptide is the only proteolytic productwe observe at the N517 N-glycosylation site , but we observe semispecificproteolytic cleavage (peptides AFINTF, KAFITNF) near the N81 site
The observation of multiple proteolytic products at site N81 may limitour ability to detect minor glycoforms
However, in trypsin- GluC digestswe observe only one proteolytic product containing site N207 , andin trypsin-chymotrypsin digests we only observe one proteolytic productcontaining site N577
Even at these sites we observe fewer glycoformsthan at N517 , which suggests that the site-specific differences inmicroheterogeneity are likely of biological or structural origin
However, we cannot rule out the possibility that differences in peptideionization may also play a role in these observations
Serum-derived ITIH4 N-linked glycopeptides demonstrate higher levels of sialylation,lower levels of fucosylation, differences in fucose linkage, and lessmicroheterogeneity compared to recombinant ITIH4
Complex, sialylatedN-glycans are present at all four canonical N-glycosylation sitesof serum-derived ITIH4
MALDI-analysis confirmed the predominanceof sialylated N-linked glycans and also corroborated the presenceof minor fucosylated glycoforms
On the basis of our site-specificcharacterization of glycopeptides from serum ITIH4 , it appears thatmost fucosylated glycoforms are restricted to sites N517 and N577 ,although we also detected a triantennary fucosylated N-glycan at N81after treatment with a broad-specificity neuraminidase
We also detectedmore branching (tri- and tetra-antennary glycoforms) at N517 and N577than at N81 and N207 , a pattern that is consistent with recombinant ITIH4
This is consistent with the fact that site specificity of N-glycanstructures, including branching, depends on the structure of the matureprotein
Processing of N-linked glycans by exoglycosidasesand glycosyltransferases, including extension and capping, take placeprimarily in the Golgi apparatus after the protein is already folded;access of glycosyltransferases to N-linked glycans is thought to beinfluenced by the protein structure
Therefore,processes governed by protein structure should have similar outcomesin recombinant and serum-derived ITIH4 if the enzymes are presentin active form
This is consistent with our findings of site-specificN-glycan branching and suggests that activity of neuraminidases islower and activity of FUT8 is higher in the HEK293 compared to humanliver, the major source of the serum derived ITIH4
This conclusionhas to be viewed, however, with caution given many unknown factorsincluding distribution and stability of the glycoforms invivo
Section : Detection of Non-Canonical N-Glycosylationat N274
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A small percentage of peptides (<1%) containingthe noncanonical N-glycosylation site NVV at N274 were occupied inboth serum and recombinant ITIH4
Noncanonical N-glycosylation hasrecently been described in immunoglobulins and a recent report alsodescribes high-mannose glycoforms at noncanonical N-glycosylationsites on several murine proteins
Using CID MS/MS, we found high-mannoseN-glycopeptides on the NVVFVIDK peptide in both serum and cell-derived ITIH4
As we find only high-mannose N-glycans at this site, its accessibilityto glycosidases and glycosyltransferases during protein maturationin the Golgi may be limited
The N274site is near the beginning of the von Willebrand factor type A ( vWF ) domain of ITIH4 , which spans residues 272–432
Protein vWFdomains are typically involved in protein–protein interactions,and glycosylation at this site could potentially affect ITIH4 interactionswith other proteins
Section : Newly Described O-Glycopeptides of ITIH4
Content :
Both recombinant and serum ITIH4 are glycosylated on peptides IPKPEASFSPRand ASFSPR containing serine residues S640 and S642
Peptides LAILPASAPPATSNPDPAVSR and LAILPASATPATSNPDPAVSR from two ITIH4 variants are glycosylatedin serum and have similar associated glycan com positions
The presenceof an additional threonine in the variant form does not impact theglycan com positions associated with the peptide
In recombinant ITIH4 ,LAILPASATPATSNPDPAVSR contains smaller andless sialylated glycan com positions compared to serum ITIH4
PeptideGPDVLTATVSGK is glycosylated exclusively in recombinant ITIH4
Initiation of O-GalNAc glycosylation through the transfer ofGalNAc to serine or threonine residues is controlled by a family ofat least 20 UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts).
Differences in glycosylation site- and tissue-specificity are expectedto be more pronounced for O-GalNAc glycosylation compared to N-glycosylation
Our observation of site-specific differences in O-glycosylation betweenserum-derived and recombinant ITIH4 is consistent with this observation
We observe the same glycan com positions(HexNAc1Hex1NeuAc1 and HexNAc1Hex1NeuAc2) on peptides IPKPEASFSPR andASFSPR in serum and recombinant ITIH4
In serum ITIH4 , we detectedglycosylation at S640 but not at S642
Glycosylation of this peptidein different contexts suggests that glycosylation at this site mayplay an important role in ITIH4 stability or function
Serum ITIH4 peptide LAILPASAPPATSNPDPAVSR ( residues691–710 ) is O-glycosylated at three sites , on residues S696,T701, and S709, and we have characterized multiple glycoforms of thispeptide
Our glycopeptide data indicate that this peptide containsglycan com positions as large as HexNAc3Hex3NeuAc4
When we analyzed detached O-glycans in serumand recombinant ITIH4 , we observed HexNAc1Hex1NeuAc1 and HexNAc1Hex1NeuAc2, and additional low-abundance glycoforms
Together, this suggests that S696, T701, and S709 are occupied withsimple core-1 O-glycans
While we do not observe this peptide in recombinant ITIH4 , we detect the PAVSRVMNMK (residues 706–715)+ HexNAc1Hex1NeuAc2glycopeptide (containing S709) in trypsin-chymo trypsin digests of recombinant ITIH4
This suggests that S709 is glycosylated in both serum and recombinant ITIH4 , while additional glycosylation occurs in serum ITIH4
We alsoobserve multiple glycoforms of peptide GPDVLTATVSGK (withpotentially glycosylated residues T506, T508, and S510) in recombinant ITIH4 , but not in serum-derived ITIH4 , and we detected two fucosylatedO-glycopeptides on GPDVLTATVSGK from recombinant ITIH4but did not detect any fucosylated O-glycopeptides in serum-derived ITIH4
In addition to the glycopeptides discussed above, evidenceof ITIH4 glycosylation on residues 719–725 has been reportedin a study of glycopeptides from urinary protein digests
However, we do not observe any glycopeptidesat this site ; this may be due to the different sources of ITIH4 orpossibly reflect proteolytic resistance of the ITIH4 sequence
Itis also possible that high levels of O-glycosylation in this regionof ITIH4 may protect ITIH4 from proteolysis in its native environmentbut could also prevent us from observing glycopeptides if high-densityglycosylation blocks access of trypsin , endoproteinase Glu-C, andchymo trypsin to proteolytic sites in this region
The ITIH4 sequenceis atypical in that it contains regions without frequent sites forcleavage of trypsin , chymo trypsin , and other common proteases
Thismakes bottom-up analyses challenging and necessitates the use of multipleproteases for observation of different glycopeptides
We detectedseveral glycoforms of glycopeptide IPKPEASFSPR in serumand recombinant ITIH4 , and we have confirmed that S640 is glycosylatedin serum ITIH4
This sequence is specific to ITIH4 isoform 1 1 and thereforecontributes to isoform-specific glycosylation that may influence ITIH4stability or interaction with the ECM
On the basis of expressionstudies, ITIH4 is primarily synthesized in the liver, and four isoforms of ITIH4 have been predicted based onmRNA and/or cDNA sequencing experiments
Three ITIH4 isoforms have been describedin adult liver tissue, while the fourth was found in fetal human liver
Isoform 1 was the first to be described and has been selected as the“canonical” sequence in UniProt
In this discussion,all references to amino acid residue locations are made in referenceto ITIH4 isoform 1 1
Isoforms 2–4 are missing small regions(all in the C-terminal half of the protein) compared to isoform 1 1
Isoform 2 also has a unique sequence “ACPSCSRSRAPAVPA”starting at residue 727
Specifically , isoforms 2 –4 do notcontain residues 621–650 of ITIH4 isoform 1 1 , and intriguingly,our proteomic analyses of serum-derived ITIH4 show that the peptideIPKPEASFSPR (and ASFSPR withone missed GluC cleavage at E) from this region of ITIH4 is O-glycosylated
Indeed , glycoprotein interactions with the ECM are frequently modulatedby the presence of O-glycans and isoform-specific O-glycosylationcould have important implications for ECM stability
We have alsoselected ITIH4 isoform 1 1 for overexpression in HEK293 cells and confirmthat this region is glycosylated in the cell line as well, and identicalglycan com positions are present in serum and recombinant ITIH4
In addition to detecting isoform-specific glycosylation, we alsodetected several O-glycopeptides that flank the proline-rich regionof ITIH4 , which may also have an impact on ITIH4 stability or interactionswith other proteins , or the ECM
ITIH4 can undergo cleavage by plasmakallikrein between R688 and R689, andmay undergo additional proteolytic degradation after this initialcleavage based on detection of native peptides in serum and plasma
ASFSPR (residues 639–644) and LAILPASA(P/T)PATSNPDPAVSR(residues 690–710), both O-glycopeptides described in our currentstudy, flank the plasma kallikrein cleavage site and the potentiallybioactive peptide
Due to the potential of glycosylation to impactproteolytic processing, these findings deserve further investigation