Glycosylations should not be neglected for the correct calculation of the molecular mass, the isoelectric point and the mass-specific UV absorption coefficient. Therefore Prot pi provides a tool to draw glycans as a posttranslational modification of proteins. This short guide deals with how to add two complex-type N-Glycosylation G1 with a sialic acid (N-acetylneuraminic acid) to the amino acid sequence of the heavy chains (HC) of a monoclonal antibody. Even though, in most cases, immunoglobulin G (IgG) is additionally modified, no further posttranslational modifications were described in this article.

How-to

First of all go to the Protein Tool of Prot pi and enter the amino acid sequence of your protein. For this tutorial the sequence of Canakinumab, a recombinant human anti-human-IL-1β IgG, from DrugBank.ca was used. The IgG is composed of four subunits – two identical heavy chains (HC) and two identical light chains (LC). Therefore add three subunits to a total of four subunits by clicking three times the “add” button (figure 1).

Enter the headline (starting with a “>”) into the first and the second text area followed by a “return”. The headlines are optional, but are helpful for the orientation in the results. Then copy the amino acid sequence of the HC on a new line below the headline in the first and the second text area.

Heavy chain:

>8836_H|canakinumab|Homo sapiens||H-GAMMA-1 (VH(1-118)+CH1(119-216)+HINGE-REGION(217-231)+CH2(232-341)+CH3(342-448))|||||||448||||MW 49253.6|MW 49253.6|
QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYY
ADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain:

>8836_L|canakinumab|Homo sapiens||L-KAPPA (V-KAPPA(1-107)+C-KAPPA(108-214))|||||||214||||MW 23357.9|MW 23357.9|
QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYY
ADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPS
RFSGSGSGTDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Copy the headline and the amino acid sequence of the LC in the same matter into the third and the fourth text area (figure 2).

Now the glycosylation is being defined. Choose “N-Glycosylation” within the “Modifications” tab and press the button “Add Modification” (figure 3).

A new window is popped up where you can specify the glycosylation (figure 4). As only the asparagine at the position 298 on the HC is glycosylated, select “Partly” option. Now you can enter the position (298) and the subunit (1) into the appropriate box.

Note that at this point only the glycosylation of one subunit can be defined. An additional modification must be added later for the glycosylation of the second subunit.

Click the “G1” button (1) in the category “Common” and a complex type G1 glycosylation appears in the field below. Then select the “NeuAc” button (2) in the “Acidic Sugar” category. Now an N-acetylneuraminic acid will be appended to the monosaccharide that you click. So click the leftmost galactose (yellow circle) of the glycan tree (3) to get a G1NeuAc glycan. Apply this glycosylation to the N(298) of the HC1 by pressing the “Save Modification” button (4).

Add now the glycosylation of the second HC in exactly the same manner but for subunit 2. After saving the second glycosylation, the table in the tab “Modifications” should look this way:

With pressing the “Calculate” button you can start the calculation of the physico-chemical parameters of the glycosylated IgG. All physico-chemical parameters are now calculated taking account of the defined N-glycosylation.

Note: Cysteine side chain sulfhydryl groups mostly form disulfide bonds in monoclonal antibodies. And in IgG, normally, the C-terminal lysine of the HC is cleaved during production and N-terminal glutamine appears as pyroglutamic acid. Therefore these modifications must be defined additionally for a correct calculation. Although this is not described in this article, further modifications can be applied in the same manner as the N-glycosylation.

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