Abstract
HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.
Highlights
Oligosaccharides expressed on glycoproteins and glycolipids are biosynthesized by glycosyltransferases in a stepwise manner
These results suggest the possibility that the Human natural killer-1 (HNK-1) carbohydrate biosynthetic key enzymes, glucuronyltransferases (GlcAT-P and GlcAT-S) and sulfotransferase (HNK-1ST), associate with each other, which results in efficient sequential biosynthesis
To investigate the interaction between the glucuronyltransferases (GlcAT-P and GlcAT-S) and HNK-1ST, FLAG-tagged glucuronyltransferases (FLAG tag fused at N terminus of each glucuronyltransferase, FLAG-P or FLAG-S) and EGFP-tagged HNK-1ST (EGFP fused at C terminus of HNK-1ST, ST-EGFP) were transiently expressed in CHO cells
Summary
Monoclonal antibody (mAb) M6749 was a generous gift from Dr H. Anti-HNK-1 mAb was purchased from the American Type Culture Collection. Mouse anti-FLAG M2 mAb and rabbit anti-FLAG polyclonal antibodies (pAb) were purchased from Sigma. Mouse anti-EGFP mAb and rabbit anti-EGFP pAb were purchased from Clontech. HRP-conjugated anti-mouse IgG and HRPconjugated anti-mouse IgM were purchased from Zymed Laboratories Inc. Alexa Fluor 568 anti-mouse IgG was purchased from Molecular. [35S]Adenosine 3Ј-phosphate,5Ј-phosphosulfate was purchased from PerkinElmer Life Sciences. Expression vectors p3XFLAG-CMV-10 and p3XFLAG-CMV-14 were from Sigma, and pEGFP-N1 and pIRES were from Clontech. PGIR201protA was kindly provided by Dr H. Rat HNK-1ST cDNA cloned into pBluescript (pBluescript/HNK1ST) was kindly provided by Dr Hans Bakker (Swiss Federal Institute of Technology). Mouse C4ST1 and human GalNAc4ST1 cDNA were kindly provided by Dr O.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
