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