NetNGlyc: Notes on the prediction output

# Predictions for N-Glycosylation sites in 1 sequence Name: CBG_HUMAN Length: 405
(Sequence) Asn-Xaa-Ser/Thr sequons (including Asn-Pro-Ser/Thr) are shown in blue. Asparagines predicted to be N-glycosylated are shown in red. Note that not all sequons are predicted glycosylated.
MPLLLYTCLLWLPTSGLWTVQAMDPNAAYVNMSNHHRGLASANVDFAFSLYKHLVALSPKKNIFISPVSISMALAMLSLG 80 TCGHTRAQLLQGLGFNLTERSETEIHQGFQHLHQLFAKSDTSLEMTMGNALFLDGSLELLESFSADIKHYYESEVLAMNF 160 QDWATASRQINSYVKNKTQGKIVDLFSGLDSPAILVLVNYIFFKGTWTQPFDLASTREENFYVDETTVVKVPMMLQSSTI 240 SYLHDSELPCQLVQMNYVGNGTVFFILPDKGKMNTVIAALSRDTINRWSAGLTSSQVDLYIPKVTISGVYDLGDVLEEMG 320 IADLFTNQANFSRITQDAQLKSSKVVHKAVLQLNEEGVDTAGSTGVTLNLTSKPIILRFNQPFIIMIFDHFTWSSLFLAR 400 VMNPV (Annotation line) `N' represents a predicted N-glycosylation site. `n' represents an Asn with a positive score, but not occuring within an Asn-Xaa-Ser/Thr sequon
..............................N................................................. 80 ...............N................................................................ 160 ................................................................................ 240 ...................N............................................................ 320 ................................................N............................... 400 ..... (Threshold=0.5) -------------------------------------------------------------------------------- SeqName Position Potential Jury NGlyc agreement result -------------------------------------------------------------------------------- CBG_HUMAN 31 NMSN 0.7166 (9/9) ++ <-- Predicted as N-glycosylated (++) CBG_HUMAN 96 NLTE 0.6356 (8/9) + <-- Predicted as N-glycosylated (+) CBG_HUMAN 176 NKTQ 0.3941 (7/9) - <-- A negative site CBG_HUMAN 260 NGTV 0.7400 (9/9) ++ CBG_HUMAN 330 NFSR 0.4223 (7/9) - see below for CBG_HUMAN 369 NLTS 0.6684 (9/9) ++ more information --------------------------------------------------------------------------------

Graphics in PostScript

The graph illustrates predicted N-glyc sites across the protein chain (x-axis represents protein length from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold (horizontal line at 0.5) is predicted glycosylated. Additional thresholds are shown at 0.32, 0.75 and 0.90 by horizontal dotted lines. Explained below. An Encapsulated postscript format of the graph is available for including in publications.

More Notes

The Asn-Xaa-Ser/Thr sequon

N-glycosylation is known to occur on Asparagines which occur in the Asn-Xaa-Ser/Thr stretch (where Xaa is any amino acid except Proline). While this consensus tripeptide (also called the N-glycosylation sequon in many texts) may be a requirement, it is not always sufficient for the Asparagine to be glycosylated. Furthermore, there are a few known instances of N-glycosylation occuring within Asn-Xaa-Cys (a Cysteine opposed to a Serine/Threonine at the N+2 position) e.g. plasma protein C (PRTC_HUMAN), von Willebrand factor (VWF_HUMAN).

NetNGlyc attempts to distinguish glycosylated sequons from non-glycosylated ones. By default, predictions are only shown on Asn-Xaa-Ser/Thr sequons. If you choose to predict on all Asparagines, then please be careful while interpreting the output. From what we know so far, only asparagines within Asn-Xaa-Ser/Thr (and in some cases, Asn-Xaa-Cys) are N-glycosylated in vivo.

In the sequence output above, Asn-Xaa-Ser/Thr sequons are highlighted in blue, and N-glycosylated Asparagines are red. With the scores for each position, Asn-Xaa-Ser/Thr sequons can be identified (in case prediction is made on all Asparagines) by a 'SEQUON' note in the right margin.

Asn-Pro-Ser/Thr

Proline just after the Asparagine, is known to preclude N-linked glycosylation in most cases by rendering the Asparagine inaccessible. NetNGlyc has been trained to ignore this Proline position (to be able to pick up other sequence signals). Thus, Asn-Pro-Ser/Thr triplets might be predicted as glycosylated but a warning is generated. Such sites may only be worth considering if there is additional confirmatory evidence.

Thresholds and confidence

Any potential crossing the default threshold of 0.5, represents a predicted glycosylated site (as long as it occurs in the required sequon Asn-Xaa-Ser/Thr without Proline at Xaa). The 'potential' score is the averaged output of nine neural networks. For further information, the jury agreement column indicates how many of the nine networks support the prediction. The N-Glyc Result column shows one of the following outputs for predictions indicating

glycosylated sites:

   + Potential > 0.5
  ++ Potential > 0.5 AND Jury agreement (9/9)  OR Potential>0.75
 +++ Potential > 0.75 AND Jury agreement
++++ Potential > 0.90 AND Jury agreement

and non-glycosylated sites:

   - Potential < 0.5
  -- Potential < 0.5 AND Jury agreement (all nine < 0.5)
 --- Potential < 0.32 AND Jury agreement

For picking up N-glycosylation sites with high specificity (Asparagines very likely to be glycosylated), use only (++) predictions (and better) for Asparagines that occur within the Asn-Xaa-Ser/Thr triplet (no Proline at the Xaa position). Note that identifying sites this way would compromise sensitivity (you may lose some positive sites).

Warnings and notes in the right margin

SEQUON ASN-XAA-SER/THR.

If you request a prediction on all Asparagines (instead of the default to predict only on Asn-Xaa-Ser/Thr sequons), then this note will appear for Asparagine positions which do occur within the Asn-Xaa-Ser/Thr sequon.

WARNING: PRO-X1.

Proline occurs just after the Asparagine residue. This makes it highly unlikely that the Asparagine is glycosylated, presumably due to conformational constraints.

WARNING: PRO-X2.

Proline occurs at the 3rd position C-terminal to the Asparagine in question (2nd 'X' in NX[ST]X). This makes it somewhat unlikely that the Asparagine is glycosylated, but this condition is not as harsh as the PRO-X1 condition.

GETTING HELP

Scientific problems: Technical problems:

Output format

More Notes

GETTING HELP