Pacific Symposium on Biocomputing 7:310-322...

Prediction of glycosylation across the human proteome and the

correlation to protein function

Ramneek Gupta and S�ren Brunak

Center for Biological Sequence Analysis� Bldg�� Bio�Centrum

Technical University of Denmark� DK�� Lyngby� Denmark�

� Introduction

The addition of a carbohydrate moeity to the side�chain of a residue in aprotein chain in�uences the physicochemical properties of the protein� Gly�cosylation is known to alter proteolytic resistance� protein solubility� stability�local structure� lifetime in circulation and immunogenicity��

Of the various forms of protein glycosylation found in eukaryotic systems�the most important types are N�linked� O�linked GalNAc �mucin�type� and O��linked GlcNAc �intracellular�nuclear� glycosylation� N�linked glycosylationis a co�translational process involving the transfer of the precursor oligosac�charide� GlcNAc�Man�Glc�� to asparagine residues in the protein chain� Theasparagine usually occurs in a sequon Asn�Xaa�Ser�Thr� where Xaa is notProline� This is however� not a speci�c consensus since not all such sequonsare modi�ed in the cell� O�linked glycosylation involves the post�translationaltransfer of an oligosaccharide to a serine or threonine residue� In this case�there is no well�de�ned motif for the acceptor site other than the near vicinityof proline and valine residues�

We have developed glycosylation site prediction methods for these threetypes of glycosylation� using arti�cial neural networks that examine correla�tions in the local sequence context and surface accessibility� In this paper� wehave used glycosylation site information on human proteins to illustrate thecontribution of glycosylation to protein function and assess how widespreadthis modi�cation is across the human proteome�

� Methods

�� Data set

Analysis shown in this paper was derived on a set of human proteins obtainedfrom the swiss�prot �rel� � database� This consisted of �� well anno�tated proteins� We chose to work with proteins from a single organism i�e�humans� to restrict the diversity of oligosaccharyltransferase acceptor sites�

Pacific Symposium on Biocomputing 7:310-322 (2002)

Glycosylation in simple organisms� such as yeast� is well studied �� but theirglycans are usually high mannosylated structures� and it is not clear how sim�ilar their mechanism of glycosylation is to that of humans� Combining datafrom di�erent organisms would complicate the analysis� possible �families� ofacceptor speci�cities causing ambiguity in distinguishing acceptor �positive�sites from non�acceptor �negative� sites�

�� Functional categories for proteins

De�ning protein function is a complicated task� and there are many di�erentways of describing the roles and functions of a protein in a cell� This is thetopic of many on�going ontology projects�� Here we chose to use a cellular roledescriptor and subcellular location as our categorisations�

�� categories �� de�ned� � unknown� re�ective of the �cellular role� of theprotein in the cell were employed �as shown in Figure �� The automatic classassignment to sequences was made by an extension of the Euclid system per�forming a lingustic analysis and clustering of swiss�prot keywords �� Key�words were parsed for the human proteins in swiss�prot� For each functionalclass� the informative weight �Z�score� of each keyword was extracted from adictionary �� Keyword sums gaves scores to all categories for a particular se�quence� The central point of the Euclid system is the dictionary� The primaryversion of this dictionary was generated from an initial set of carefully� handannotated proteins from di�erent organisms spanning every kingdom of life�From this initial set� a �rst dictionary was de�ned which was used to assign allswiss�prot proteins and the process of dictionary de�nition and assignmentwas reiterated until convergence� This �nal dictionary obtained was used toassign functional classes to around �� human proteins from swiss�prot�

The cellular role categories themselves were derived from an earlier pro�posed scheme for Escherichia coli � which was later extended by the TIGRgroup for other complete genomes� These categories comprise � functionalclasses which are subsets of three superclasses� Energy� Communication andInformation� Proteins which do not �t in the � categories are assigned to�Other� �functionally unde�ned cluster� or to �Unknown� �sequences which donot contain the relevant keywords needed for classi�cation in the above sys�tem��

Subcellular locations of proteins were obtained from swiss�prot annota�tions and psort predictions� �where no parsable swiss�prot annotation wasfound��


� Results

�� N�Glycosylation

N�linked glycosylation modi�es membrane and secreted proteins� This co�translational process occurs in the endoplasmic reticulum and is known toin�uence protein folding� The modi�cation attributes various functional prop�erties to a protein� To examine if certain categories of proteins were more proneto glycosylation than others� we studied the spread of known glycosylation sitesacross di�erent categories�

N�glycosylation may also display some positional preferences in the proteinchain� Speci�cally� it has been shown that sites need to be �� residues awayfrom the N�terminus �� and that glycosylation e�ciency is reduced within ��residues of the C�terminus ��

In our data set of approximately �� human proteins� only � proteins�at �� con�rmed sites� were annotated in swiss�prot as N�glycosylated �notconsidering proteins with only potential or probable sites�� Figure � il�lustrates the spread of human glycosylation sites along the protein chain andacross predicted subcellular locations and keyword based assignment of cel�lular role categories� Relative positions of sites on proteins were calculatedwith respect to normalised sequence lengths� The sequence length� dividedinto tenths is shown along the x�axis� from the N�terminal start on the left tothe C�terminal end on the right�

N�glycosylated proteins appeared to almost exclusively belong to the func�tional category� �Transport and binding�� This may not be too surprising con�sidering that this category consists largely of membrane and secreted proteins�Only a few proteins belonged to any other cellular role category and most ofthese appeared involved in central intermediary metabolism� Subcellularly� ex�tracellular proteins were the most favoured and others occurred in membraneproteins and in the endoplasmic reticulum or Golgi�

A clear positional preference for glycosylation sites on protein chains wasapparent� The terminal ends of proteins seemed unfavourable and most sitesseemed to occur N�terminal to the centre of the protein chain �� to �� alongthe length from the N�terminal start�� The frequency of sites smoothly taperedo� on both ends from this peak with a longer C�terminal tail� This statisticalobservation agrees with speci�c experimental indications of a �� residuedistance from the N�terminal and a �� residue distance from the C�terminalend �� One peculiar observation from the �gure was the C�terminal sites innuclear proteins� On examination� these turned out to be around �� proteinswhich were indeed annotated to be N�glycosylated in the C�terminal� However�this seems to be an anomaly of the sub�cellular prediction by psort� For


N−Glyc site positions across subcellular compartments

Relative position across protein chain −−>Nterm

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N−Glyc site positions across cellular role categories

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Amino acid biosynthesis

Biosynthesis of cofactors

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Central intermediary metabolism

Energy metabolism

Fatty acid and phospholipid metabolism

Other categories

Purines and pyrimidines

Regulatory functions

Replication

Transcription

Translation

Transport and binding proteins

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Figure �� Categorical distribution of known N�glycosylation sites across the pro�tein chain� Colour indicates frequency of sites �green to pink in increasing order�� Pro�tein chains� normalised in length� are represented across the x�axis from N�terminal to C�terminal� Subcellular locations �top� were predicted using psort� and cellular role classi��cation �bottom� by lexical analysis of swiss�prot keywords �Alfonso Valencia et al�� MostN�glycosylation sites were clustered in the �rst half of all protein chains� and mainly occurredin extracellular transport and binding proteins�


instance� some secreted proteins among these were Vasopressin�Neurophysin��Copeptin precursor� Von Willebrand Factor Precursor and ImmunoglobulinDelta Chain C�

Experimental determination of glycosylation sites is di�cult to achieve aslarge amounts of puri�ed protein are needed for the analysis of glycosylationsites� In addition� glycosylation can be an organism� and tissue speci�c event�Therefore only a few glycoproteins have been characterised so far as re�ectedin the low percentage of glycoprotein entries in swiss�prot �approx� �� ofhuman proteins� see also �� This motivates the need for developing theoreticalmeans of predicting the glycosylation potential of sequons�

�� O�linked GalNAc Glycosylation

The addition of GalNAc linked to serine or threonine residues of secreted andcell surface proteins� and further addition of Gal�GalNAc�GlcNAc residues ��is also known as mucin type glycosylation and is catalysed by a family ofUDP�N�acetylgalactosamine� polypeptide N�acetylgalactosaminyltransferases�GalNAc�transferases�� The modi�cation� a post�translational event� takesplace in the cis�Golgi compartment�� after N�glycosylation and folding of theprotein� and a�ects secreted and membrane bound proteins�

There is no acceptor motif de�ned for O�linked glycosylation� The onlycommon characteristic among most O�glycosylation sites is that they occur onserine and threonine residues in close vicinity to proline residues� and that theacceptor site is usually in a beta�conformation� A prediction method �� forthis type of glycosylation on mammalian proteins has been built earlier andmade available as a web server a� A database of O�glycosylated sequences isalso availableb and was used in constructing the O�glycosylation site predictionmethods ��

Figure � shows the spread of predicted glycosylation sites �O�GalNAc�mucin�type� across di�erent categories and across the protein chain� To con�struct this plot� sequence lengths were normalised� and relative position ex�pressed on a percent �� scale� Glycosylation sites were binned �� binsacross each sequence�� and their frequency plotted across di�erent categories�Sites tend to cluster towards the C� and N�termini of proteins for some cat�egories� This �gure also shows that O�glycosylation acceptor sites occur ina wide range of proteins� though glycosylation patterns �frequency� positionsacross chain� may di�er for di�erent types of proteins�

ahttp�www�cbs�dtu�dkservicesNetOGlycbhttp�www�cbs�dtu�dkdatabasesOGLYCBASE


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Figure � Postional O�GalNAc glycosylation� O�GalNAc �mucin type� glycosylationdisplays preference for position across a protein chain which could be signi�cant acrossdi�erent categories� The Position axis re�ects normalised protein chain length from N�terminal � on the axis� to C�terminal �� The height of the bars indicates the numberof predicted O�GalNAc sites �in � �� human proteins� for a particular category in aparticular position bin�


�� O�linked GlcNAc Glycosylation

Glycosylation of cytosolic and nuclear proteins by single N �acetylglucosamine�GlcNAc� monosaccharides is known to be highly dynamic and occurs on pro�teins with wide�ranging functions and cellular roles ��N �acetylglucosamine�donated by the nucleotide precursor UDP�N �acetylglucosamine� is attached ina beta�anomeric linkage to the hydroxyl group of serine or threonine residues�

So far� all proteins with O��GlcNAc linked residues� are also known to bephosphorylated� Evidence suggests that at least in some cases� these two post�translational modi�cation events may share a reciprocal relationship�� Thispeculiar behaviour strongly suggests a regulatory role for this modi�cation�Sites which can be both glycosylated and alternatively phosphorylated arealso known as �yin�yang� sites ��

The acceptor site for O��GlcNAc glycosylation does not display a de�niteconsensus sequence� nor are there many annotated sites in public databases�However� the fuzzy motif is marked by the close proximity of Proline andValine residues� a downstream tract of Serines and an absence of Leucine andGlutamine residues in the near vicinity �data not shown�� A prediction methodfor this type of glycosylation on human proteins has been built and madeavailable c as a web server �in preparation��

Out of approximately �� human sequences from swiss�prot �rel� ��over �� had at least one predicted O�GlcNAc site� �� of these proteinshad at least one high scoring O�GlcNAc site prediction �with �� high scoringSer�Thr sites�� A number of these were DNA�binding proteins and involved intranscriptional regulation� When ranked according to scores� a large fraction atthe top of this list were found to be nuclear proteins �as annotated in swiss�

prot�� The O�GlcNAc transferase itself �P�� subunit� was found to havepredicted O�GlcNAc sites�

To study if the O��GlcNAc modi�cation was speci�c for certain types ofproteins� we classi�ed the potentially modi�ed proteins into cellular role cat�egories and subcellular locations� Figure illustrates the spread of proteinswith at least one high�scoring O��GlcNAc site� across di�erent categories�Also shown in this �gure is the spread of phosphorylated proteins �as pre�dicted�� by NetPhosd�� Yin�yang� proteins� proteins with pest regions�� andproteins with O��GlcNAc �� sites which fall within pest regions�

chttp�www�cbs�dtu�dkservicesYinOYangdhttp�www�cbs�dtu�dkservicesNetPhos


pest-glcnac

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Distribution of sites across categories of (swissprot) human proteins

Subcellular locations

Cellular role categoriesCellular role categories for proteins were predictedusing a linguistic approach (on keywords) by Valencia et al.SWISS-PROT

Subcellular locations for protein wereobtained from a combination of annotations and predictions.SWISS-PROT PSORT

Figure �� Predicted O��GlcNAc sites across the human proteome� The two panels�top� bottom� indicate di�erent categorisations of proteins as depicted in the innermost andoutmost circles of the pies� Individual rings represent di�erent post�translational modi��cations and their occurrence in the corresponding category� E�g�� phosphorylation occurswidely across all categories of proteins� Potential O�GlcNAc sites occur in half of all nuclearproteins and regulatory proteins� They also occur widely in replication and transcriptionproteins� Proteins with pest regions and O�GlcNAc sites are mostly regulatory althoughpest regions themselves also occur in other categories�


While the O��GlcNAc modi�cation seems to potentially a�ect almostall types of proteins� most O�GlcNAcylated proteins were either regulatoryproteins or �transport and binding� proteins� A large fraction of unclassi�edproteins ��unknown� in role categories� were also predicted to contain this modi��cation� Over half of all nuclear proteins contained a high ranking O��GlcNAcmodi�ed site� Cytoplasmic proteins� membrane proteins and secreted proteinsalso contained potential sites�

Phosphorylation is a very wide�spread modi�cation �� This is re�ected inour graphs as phosphorylation sites �� potential by NetPhos� appeared wellrepresented in all protein categories� However� Yin�yang sites appeared to existlargely in regulatory proteins� transcription related proteins or �transport andbinding proteins�� and were mostly nuclear� O�GlcNAcylated pest regions werealso mostly nuclear� though a large membrane fraction also existed� Aroundhalf of all these proteins were involved in regulatory functions�

In an additional study� the number of potential O��GlcNAc sites in pro�teins was studied with respect to function and cellular location� Figure � illus�trates the number of predicted �high�scoring� sites per �� Ser�Thr residues�per protein�� Proteins with �� predicted GlcNAc sites �per �� Ser�Thr�were predominantly nuclear� cytoplasmic or membrane proteins� Nuclear andcytoplasmic proteins carried the highest densities of sites� a few cytoplasmicproteins having as many as �� high�scoring O�GlcNAc sites among �� Ser�Thrresidues� With respect to cellular roles� proteins belonging to the category�Purines� pyrimidines� nucleosides and nucleotides� contained well spaced outsites �only a few sites among �� Ser�Thr residues�� Proteins with a widerdistribution of sites included regulatory� transcription� replication� �transportand binding�� cell envelope and the �unknown� category proteins� The high�est density of sites �� per �� Ser�Thr� was found in transcription andregulatory proteins� though some �unknown� proteins had over �� sites �per�� Ser�Thr�� In general� the intracellular O��GlcNAc modi�cation does notseem to cluster among close residues or display any characteristic spacing aswas evident for the O��GlcNAc modi�cation a�ecting surface and membraneproteins of Dictyostelium discoideum ��


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Figure �� Number of predicted O��GlcNAc sites per �� Ser�Thr� in di�erentcategories of human proteins� �A� shows proteins in di�erent subcellular locations and�B� indicates cellular role categories� The z�scale � �� in A or �� in B� is a frequencycount for a particular bin� e�g� � O�GlcNAcs �per � SerThr� occur most frequently fornuclear proteins in �A�� These modi�cations usually do not occur in clusters� Although po�tential acceptor sites are largely found in nuclearcytoplasmic proteins �usually regulatory��they also surprisingly occur in membrane proteins �mostly transport and binding proteins��


Human proteome�wide scans revealed that the O��GlcNAc acceptor pat�tern occurs across a wide range of functional categories and subcellular com�partments� For humans� the most populated functional categories were regu�latory proteins and transport and binding proteins� Nuclear and cytoplasmicproteins were prominent� though membrane and secreted proteins were sur�prisingly also in high numbers� It is interesting to know that acceptor patternsexist on these proteins too� but the cellular machinery de�nes protein target�ting and consequently in�uences their modi�cations� The prediction serverguards against this possibility by generating a warning when a potential signalpeptide is detected by SignalP e�

PEST regions� rich in the amino acids Proline �P�� Glutamic acid �E��Serine �S� and Threonine �T�� are hypothesised to be degradative signals forconstitutive of conditional protein degradation�� Phosphorylation� a commonmechanism to activate the pest�mediated degradation pathway� may be sig�nalled by deglycosylation in the same region� Our scans revealed that a smallfraction of O�GlcNAc sites appeared in PEST regions� Such sites were mostlyfound in proteins involved in regulatory functions�

� Final Remarks

Glycosylation is clearly a modi�cation a�ecting a wide range of proteins� and isnow known to a�ect both intracellular and secreted proteins� Di�erent types ofglycosylation have varying site preferences on proteins� and occur in di�erentpatterns across the protein chain�

In a project �in preparation� predicting protein function solely from pro�tein chain global properties �molecular weight� length� etc�� and potentialpost�translational modi�cations� glycosylation was one of the most importantdeterminants for functional classi�cation�

Since characterising glycoproteins experimentally is a tedious and time�consuming task� it is worthwhile at this juncture to develop tools for predict�ing glycosylation sites� This is essential information for deciphering proteinfunction and characterising complete proteomes�

� Acknowledgements

The Danish National Research Foundation is acknowledged for support�

ehttp�www�cbs�dtu�dkservicesSignalP


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