Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [1] Polypeptide N-acetylgalactosaminyltransferase family
Pp-GalNAc-Ts are the initiation enzyme to synthesize the mucin-type O-glycan, which catalyze the transfer of GalNAc from UDP-GalNAc, a donor substrate, to Ser/Thr residue of polypeptide chain, an acceptor substrate. A method to assay the enzymatic activity and to analyze the reaction products from a synthesized peptide as a substrate by HPLC system is described here.
UDP-GalNAc as a donor substrate (Sigma-Aldrich, St. Louis, MO)
Peptide substrate as an acceptor substrate
TALON metal affinity resin (Takara Bio Inc., Otsu, Japan)
Superdex 200 10/300 GL column (GE Healthcare, Little Chalfont, UK)
C18 reverse phase column (Nacalai/Cosmosil: 5C18-AR, 4.6 × 250 mm)
HPLC system
Ultra-filtration system for large volume (>1 L)
Enzyme reaction
The reaction mixture contains, 25 mM Tris-HCl (pH 7.4), 5–10 mM MnCl2, 0.1% Triton X-100 (optional), the donor substrate UDP-GalNAc, the acceptor peptide and the enzyme. The total volume is 20–50 μL. If a crude enzyme source is used, proteinase inhibitor cocktail and/or PMSF could also be included to prevent from degradation of the substrate peptide.
Concentrations of the substrates should be optimized. A possible combination is 250 μM UDP-GalNAc and 150 μM acceptor peptide. The later depends on the detection method. The amount of enzyme should also be optimized. The enzyme activity will be various upon the peptide substrate. A typical specific activity is 10–100 μmol/h/mg protein, when the both substrates are sufficient.
The reaction is initiated by addition of the enzyme solution or UDP-GalNAc into the reaction mixture.
The reaction mixture is incubated at 37°C for 15 min to 24 h, which should be modified for the experimental purpose. If the reaction mixture is a suspension, the tube should be agitated through the reaction time.
The reaction is stopped by the boil at 98°C for 3 min or addition of 0.1% (final concentration) TFA.
Analysis
Please see Section [VI] “General methods for detection of enzyme reaction products” .
Note for how to prepare enzyme as a recombinant protein. 1) Design the expression construct. The concepts below are probably shared among the other glycosyltransferase families belonging into the type-II membrane protein. 1.1) Truncation site. A soluble truncated form of the pp-GalNAc-T enzyme is suitable for this protocol. A possible truncation site is just after the N-terminal hydrophobic transmembrane stretch and before the Cys residue in the catalytic domain (probably the first Cys from the N-terminus). The catalytic domain must be integral. A sequence alignment analysis is useful to predict the catalytic domain. 1.2) Purification tag. Introduction of some purification and/or detection tag(s) is useful to check the expression level and to purify the expressed protein. Hexa-His tag and FLAG tag (Sigma) are well used. Various tags, such as protein domain, HA, MBD, are also available. An appropriate tag suitable for available instruments can be selected. The site of the tag will be just after the truncation site. Unfolded linker sequence will be acceptable without obvious interference on the enzymatic activity. 1.3) Secretion tag. A soluble truncated protein is easily secreted into the culture medium by addition of a secretion tag, such as preprotrypsin signal sequence. 1.4) Expression host. The designated DNA construct will be inserted into the expression vector and be transformed into the host cells. For example, pPIC9 (Invitrogen) is one of the most used vectors for P. pastoris. pFLAG-CMV is popular for expression with mammalian culture cells. The selection of the expression host is one of the most influential factors to express the human glycosyltransferases. As for pp-GalNAc-T, yeast is considered a suitable host, at the points of yield, cost, biochemical handling and bacteria-like culturing procedure. Alternatively, mammalian cells and insect cells are also useful to express the active enzymes. On the contrary, bacterial cells and the in-vitro translation systems are not successful without extensive modifications, such as a refolding process. 2) Expression. The recombinant enzyme source of pp-GalNAc-T can typically be expressed in large scale by yeast expression system. The methylotrophic yeast, P. pastoris, is preferably used. 3) Purification. The purification of the enzyme source is not necessary; however, it is desirable to avoid possible artificial troubles. Purification protocol depends on the expression construct.
Note for peptide substrate. The substrate specificity of pp-GalNAc-T restricts the peptide sequence of the substrate. The design of the sequence should be well considered. In a pilot study, Muc1a (AHGVTSAPDTR), Muc2 (PTTTPISTTTMVTPTPTPTC) and/or Muc5AC (GTTPSPVPTTSTTSAP) are typically used. These peptide substrates can be synthesized with a peptide synthesizer. Commercial synthesis service is also available. A fluorescence group is very useful to increase the detection signal on HPLC analysis. The group can be attached at N- or C-terminus. (Note that the modification of the N-terminus could interfere with the Edman degradation of the peptide sequence analysis.) An artificial, additional Lys residue can be attached to introduce the fluorescence tag. Some pp-GalNAc-Ts require a prior GalNAc on the peptide substrate. In this case, GalNAc-attached peptide must be prepared, which can be produced by another pp-GalNAc-T or by chemical synthesis with a peptide synthesizer using GalNAc-Ser/Thr. The peptide is dissolved into water, if possible, and store at −20°C without light. Prior to the enzyme assay, the HPLC elution conditions and elution pattern, and/or MS should be confirmed.
