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YinOYang - 1.2

O-(beta)-GlcNAc glycosylation and Yin-Yang sites (intracellular/nuclear proteins)

The YinOYang WWW server produces neural network predictions for O-ß-GlcNAc attachment sites in eukaryotic protein sequences. This server can also use NetPhos, to mark possible phosphorylated sites and hence identify "Yin-Yang" sites.

Submission


Sequence submission: paste the sequence(s) or upload a local file

SUBMISSION

Paste a single sequence or several sequences in FASTA format into the field below:

Submit a file in FASTA format directly from your local disk:

    generate graphics
    yin-yang site predictions (i.e. cross-NetPhos scans)

Output
show only positive sites
show all S/T residues

NetPhos threshold    

SignalP is automatically run on all sequences. A warning is displayed if a signal peptide is predicted.


Restrictions:
At most 2000 sequences and 200000 amino acids per submission; each sequence not less than 21 and not more than 4000 amino acids.

Confidentiality:
The sequences are kept confidential and will be deleted after processing.


CITATIONS

For publication of results, please cite:

Prediction of glycosylation sites in proteomes: from post-translational modifications to protein function.
R Gupta.
Ph.D. thesis at CBS, 2001.

Prediction of glycosylation across the human proteome and the correlation to protein function.
Gupta, R. and S. Brunak.
Pacific Symposium on Biocomputing, 7:310-322, 2002.

PMID: 11928486         Download the full article in PDF.


ACKNOWLEDGEMENTS

Experimental data used in training this method was provided by Gerald Hart, Lance Wells and other members of their laboratory.

The YinOYang server incorporates results from NetPhos and SignalP.

Instructions



In order to use the YinOYang WWW server for prediction on amino acid sequences:

  1. (optional) Enter a name for the sequence.

  2. Enter a single sequence (or a Swissprot ID or AC) in the sequence window. Alternatively, give a file name containing sequences in FASTA format (multiple sequences allowed).

    The sequence must be written using the one letter amino acid code: `acdefghiklmnpqrstvwy' or `ACDEFGHIKLMNPQRSTVWY'.
    Other letters will be converted to `X' and treated as unknown amino acids.
    Other characters, such as whitespace and numbers, will simply be ignored.

  3. Choose the output format: Show only positive predictions, or show potentials for all Ser/Thr sites in the sequence(s).

  4. Include Graph: A graphic illustrating glycosylation potentials across the sequence length will be generated (recommended).

  5. "Yin-yang" site predictions: Checking this box will (in parallel) generate predictions from the NetPhos server and will indicate Ser/Thr sites which have a potential for being O-GlcNAcylated as well as phosphorylated ('yin-yang' sites). The graphic is affected accordingly.

  6. NetPhos threshold: This may be adjusted (0-1, default: 0.5). Sites are indicated as phosphorylated only if their NetPhos potentials are above this fixed threshold.

  7. Press the "Submit sequence" button.

  8. A WWW page will return the results when the prediction is ready. Response time depends on system load, but is usually only a few seconds.

Output format



Name:  HXA3_HUMAN       Length:  443

(sequence)

MQKATYYDSSAIYGGYPYQAANGFAYNANQQPYPASAALGADGEYHRPACSLQSPSSAGGHPKAHELSEACLRTLSAPPS      80

QPPSLGEPPLHPPPPQAAPPAPQPPQPAPQPPAPTPAAPPPPSSASPPQNASNNPTPANAAKSPLLNSPTVAKQIFPWMK     160

ESRQNTKQKTSSSSSGESCAGDKSPPGQASSKRARTAYTSAQLVELEKEFHFNRYLCRPRRVEMANLLNLTERQIKIWFQ     240

NRRMKYKKDQKGKGMLTSSGGQSPSRSPVPPGAGGYLNSMHSLVNSVPYEPQSPPPFSKPPQGTYGLPPASYPASLPSCA     320

PPPPPQKRYTAAGAGAGGTPDYDPHAHGLQGNGSYGTPHIQGSPVFVGGSYVEPMSNSGPALFGLTHLPHAASGAMDYGG     400

AGPLGSGHHHGPGPGEPHPTYTDLTGHHPSQGRIQEAPKLTHL

(annotation line, G=O-GlcNAc, Y=YinYang)

...................................G...................GY..................G...G      80

..................................Y.......GG.Y.....G...G........................     160

.....Y...GYYY..........Y.....Y.....G..G.........................................     240

................G.Y...Y.G...........................Y....G.....G......G...G..G..     320

........................................................................G.......     400

........................................G..



-------------------------------------------------------------------------------

SeqName     Residue  O-GlcNAc  Potential  Thresh.  Thresh.   NetPhos  YinOYang?

                      result   (o-glcnac)   (1)      (2)    potential

                                                          (Thresh=0.5)

-------------------------------------------------------------------------------

HXA3_HUMAN     5  T    -        0.4047    0.4125   0.5064     <-- A negative site    

HXA3_HUMAN     9  S    -        0.4267    0.4631   0.5746            

HXA3_HUMAN    10  S    -        0.3548    0.4653   0.5776            

HXA3_HUMAN    36  S    ++       0.5781    0.4421   0.5463     <-- O-GlcNAc predicted (++)       

HXA3_HUMAN    51  S    -        0.4089    0.4299   0.5298            

HXA3_HUMAN    54  S    -        0.3183    0.3966   0.4849     0.783  <- Phos,No-GlcNAc

HXA3_HUMAN    56  S    +        0.4483    0.3792   0.4615            

HXA3_HUMAN    57  S*   +        0.4357    0.3856   0.4702     0.967       * <- YinYang

HXA3_HUMAN    68  S    -        0.2631    0.4479   0.5542            

HXA3_HUMAN    74  T    -        0.3126    0.4422   0.5464            

HXA3_HUMAN    76  S    ++       0.5023    0.3922   0.4791            

HXA3_HUMAN    80  S    +++      0.6007    0.3667   0.4447            

HXA3_HUMAN    84  S    -        0.2640    0.3769   0.4584     0.991       

HXA3_HUMAN   115  T*   +++      0.6261    0.3591   0.4344     0.770       * <- YinYang
.

.

.

-------------------------------------------------------------------------------


O-GlcNAc Result
This can be one of 5 possibles:-

   -  No O-GlcNAc predicted

O-GlcNAc predicted: (different strengths)
   +  Potential > Thresh-1
  ++  Potential > Thresh-2          (Thresh-2 is a threshold based on more stringent surface measures)
 +++  Potential > (Thresh-2 + 0.1)
++++  Potential > (Thresh-2 + 0.1) AND Potential >= 0.75

NetPhos
These potentials are displayed if 'YinYang' output was selected and if the NetPhos potential
crosses the NetPhos (fixed) threshold.
Predictions are run in parallel from the NetPhos server.

YinOYang
Ser/Thr residues which are predicted to be O-GlcNAcylated as well as phosphorylated are
marked by an asterisk (*) in both the residue and the YinOYang column. Such sites may be
reversibly and dynamically modified by O-GlcNAc or Phosphate groups at different times
in the cell.

SignalP
With all predictions, the SignalP server is run in parallel. If a sequence is predicted to contain
a signal peptide, a warning is displayed. Such sequences are unlikely to be intracellular, and hence
unlikely to be O-GlcNAcylated.

Graph
The figure illustrates O-GlcNAc and NetPhos predictions across the length of the sequence.
The x-axis represents the sequence from N-terminal to C-terminal. Vertical impulses (green)
are O-GlcNAc potentials. For predicted O-GlcNAcylated sites, these potentials would cross
the threshold (blue wavy horizontal) line.
Small red marks/circles on the green impulses indicate YinYang sites.



YinOYang Abstract


Intracellular O-glycosylation is characterised by the addition of N-acetylglucosamine, in a beta anomeric linkage, to Serine and Threonine residues in a protein. The acceptor site does not display a definite consensus sequence. However, the fuzzy motif is marked by the close vicinity of Proline residues (positions -4,-3,-2), Valines (-1,+2,+4,+5) and a downstream tract of Serines (+1,+4,+7) though Leucines and Glutamines are disfavoured. Secondary structure predictions indicate the 21-mer window to be sheet or coil. We train a jury of neural networks on 40 experimentally determined O-(beta)-GlcNAc acceptor sites, to recognise the sequence context and surface accessibility. Non-acceptor Serine/Threonines were pruned from 1251 in number to 626. In a cross-validation, 72.5% of the glycosylated sites and 79.5% of the non-glycosylated sites were correctly identified in the test set, revealing a Matthews correlation coefficient of 0.22 on the original data, and 0.84 on the augmented data set.

The method was used to scan all human protein sequences available in SwissProt for potential O-(beta)-GlcNAc acceptors. Since this modification is known to be reciprocal with phosphorylation, we cross scanned for phosphorylation sites, and identified such 'Yin-Yang' sites. The spread of O-(beta)-GlcNAcylation, PEST regions and phosphorylation sites, was studied across cellular role categories, enzyme classes and subcellular compartments. Predicted O-(beta)-GlcNAc sites were found in over half of all SwissProt human sequences, 65% of which were nuclear or cytoplasmic.

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