PMID: PMC6145689-1-6

 

    Legend: Gene, Sites

Title : Intact N-glycopeptide marker panel for PCa diagnosis

Abstract :
  1. To provide a panel of confidently characterized PCa-associated intact glycopeptides , we selected only intact N-glycopeptides with significant changes in abundance, whose glycosylation sites were also identified in the de-N-glycosylated peptide fractions and whose glycan com positions were commonly known human serum glycoforms reported in UniCarbKB [38]
  2. Besides, we considered only intact glycopeptides displaying fold changes greater than 1.3 or less than 0.77 when comparing PCa to BPH urine
  3. A total of 56 intact N-glycopeptides matched these criteria (Table 1)
  4. Information of the relative levels of their corresponding glycoproteins was obtained using quantitative LC-MS/MS data of the non-modified peptide fraction (Supplementary Table 10)
  5. Table 1 shows site-specific glycoforms that are more/less abundant in PCa independently of the protein level
  6. For example, protein AMBP AMBP ( AMBP ) was clearly more abundant at the protein level (as detected by ten non-modified peptides with increased levels, Supplementary Table 9) and site-specific glycan regulation was observed by the increased relative abundance of three intact glycopeptides covering two N-glycosylation sites in PCa urine (Table 1)
  7. On the other hand, apolipoprotein D ( APOD ) was less abundant in PCa (two non-modified peptides with decreased levels, Supplementary Table 9), but increased levels of site-specific glycosylation (i.e. HexNAc(7)Hex(6)) were observed
  8. In contrast, LTF was found to be both less abundant at the protein level (six under-represented non-modified peptides in PCa) and with respect to the N-glycosylation site occupancy as evaluated by the under-representation of the de-N-glycosylated peptide TAGWNIPMGLLFDQTGSCK and the site-specific hybrid glycoform HexNAc(4)Hex(7)NeuAc(1) in PCa
  9. Most of the glycoproteins showed no changes at the protein level, but were aberrantly changing with respect to their site-specific glycosylation levels e.g. CLU , LOX , SERPINA4 , WFDC2 , LAIR1 , PIK3IP1 (Table 1)
  10. This indicates that the glycosylation machinery, and not the protein translation and secretion, of the cells producing these cancer-associated glycoforms is significantly impacted by the malignant processes associated with PCa
  11. Some glycopeptides of the urinary glycoproteome were putatively identified with terminal NeuGc
  12. Although being a non-human type of sialic acid, NeuGc may, however, be a component of human glycoproteins arising from exogenous building blocks in particular in glycoproteins of cancer cell origin [39]
  13. These putative NeuGc-containing glycopeptides were manually checked for the presence of the corresponding oxonium ions i.e. m/z 308/290 in the corresponding HCD-MS/MS spectra
  14. All of the glycopeptides showed diagnostic ions for NeuAc (m/z 274/290) instead of NeuGc
  15. As verified by manual annotation, the NeuAc-containing glycoforms were found to be the correct glycan com position in all of these cases, Table 1
  16. Besides, the com position HexNAc(4)Hex(5)NeuGc(1) was found to be incorrectly identified due to an concomitant oxidation of the peptide
  17. In fact, the correct com position was HexNAc(4)Hex(5)NeuAc(1)
  18. PCA was performed using a panel of 56 intact N-glycopeptides identified with high confidence
  19. Complete segregation was observed between PCa and BPH (Figure 4A)
  20. Interestingly, both the abundance of the glycosylation sites (as measured by the de-N-glycosylated peptides ) and proteins (measured by the non-modified peptides ) from the glycoproteins that were present at different levels in PCa vs BHP were not able to discriminate between the two diseases (Figure 4B–4C)
  21. These results indicate that most PCa-specific glyco-features are not due to quantitative changes in the protein or site occupancy level, but arise from altered abundance of specific glycoforms of the individual urinary glycoproteins from the PCa patients
  22. The PCa-specific panel of 56 intact N-glycopeptide was also visualized by a heat map
  23. Unsupervised clustering was able to accurately separate the PCa and BPH donors groups (Figure 4D)
  24. The N-glycopeptide panel was also evaluated by ROC curve analysis
  25. The AUC was calculated using combinations of 3, 5, 10, 20, 28 and 56 selected N-glycopeptides within the entire glycopeptide panel (56 N-glycopeptides )
  26. Using 20, 28 or 56 intact N-glycopeptides , an AUC of 1 was obtained demonstrating high specificity and sensitivity of these urinary glycopeptides in the discrimination between the PCa patients from the BHP patients (Figure 4E)
  27. The relationship between the glycoproteins that carried aberrant N-glycosylation in PCa relative to BHP was explored in a protein-protein network analysis to search for over-represented biological functions and pathways
  28. Significant enrichment of pathways pertaining to the complement and coagulation cascade (KEGG pathway) and a regulation of immune system response (Gene Ontology Biological process) were observed in the network of glycoproteins carrying aberrant glycosylation in PCa (Figure 4F)
  29. In support of our finding, many of the glycoproteins in our panel were already shown in previous studies to be altered in PCa or involved in tumor growth and development
  30. For example: AMBP for PCa diagnosis [40], CD59 associated with PCa progression [41–43], CLU as a therapeutic target against PCa [44] and predictor for PCA recurrence [45], and PTGDS was found in increased concentration in urine from PCa patients [40]
  31. The association of these glycoproteins to PCa in these previous studies was established based on the protein expression level
  32. HexNAc(5)Hex(6)Fuc(1)NeuAc(2)NeuGc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(1)NeuGc(1), HexNAc(5)Hex(6)Fuc(1)NeuAc(2)NeuGc(1), HexNAc(4)Hex(5)Fuc(1)NeuAc(1)NeuGc(1) HexNAc(5)Hex(7)NeuAc(3), HexNAc(5)Hex(7)NeuAc(2), HexNAc(5)Hex(7)NeuAc(3), HexNAc(4)Hex(6)NeuAc(2), In contrast, we show here for the first time, that site-specific glycan com positions are also altered in this set of glycoproteins in PCa
Output (sent_index, trigger, protein, sugar, site):
  • 0. N-glycopeptide, , -, -, N-glycopeptide
  • 1. N-glycopeptides, , -, -, N-glycopeptides
  • 1. glycopeptides, , -, -, glycopeptides
  • 1. glycosylation, , -, -, sites
  • 11. glycopeptides, , -, -, glycopeptides
  • 12. glycoproteins, , glycoproteins, -, -
  • 12. glycoproteins, , glycoproteins, NeuGc, -
  • 13. glycopeptides, , -, -, glycopeptides
  • 14. glycopeptides, , -, -, glycopeptides
  • 15. composition, , -, all, position
  • 16. composition, , -, the composition HexNAc(4)Hex(5)NeuGc(1), position
  • 18. N-glycopeptides, , -, -, N-glycopeptides
  • 2. glycopeptides, , -, -, glycopeptides
  • 20. de-N-glycosylated, , -, -, peptides
  • 20. glycoproteins, , glycoproteins, -, -
  • 20. glycosylation, , -, -, sites
  • 21. glycoforms, , glycoproteins, -, -
  • 21. glycoproteins, , glycoproteins, -, -
  • 22. N-glycopeptide, , -, -, N-glycopeptide
  • 24. N-glycopeptide, , -, -, N-glycopeptide
  • 25. N-glycopeptides, , -, -, N-glycopeptides
  • 25. glycopeptide, , -, -, glycopeptide
  • 26. N-glycopeptides, , -, -, N-glycopeptides
  • 26. glycopeptides, , -, -, glycopeptides
  • 27. glycoproteins, , glycoproteins, -, -
  • 28. glycoproteins, , glycoproteins, -, -
  • 29. glycoproteins, , glycoproteins, -, -
  • 3. N-glycopeptides, , -, -, N-glycopeptides
  • 31. glycoproteins, , glycoproteins, -, -
  • 32. glycoproteins, , glycoproteins, -, -
  • 4. glycoproteins, , glycoproteins, -, -
  • 6. N-glycosylation, , -, -, sites
  • 6. glycopeptides, , -, -, glycopeptides
  • 8. N-glycosylation, , -, -, site
  • 9. glycoproteins, , glycoproteins, -, -
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX