**12. HNK-1 Carbohydrate Epitope as a Recognition Motif**

The human natural killer-1 (HNK-1) carbohydrate motif is a unique sulphated trisaccharide of the structure SO4-3GlcAβ1-3Galβ1-4GlcNAc, which is developmentally and spatially expressed in a cell-type specific manner within the CNS (Figure 3a–d) [207]. HNK-1 is also a well-known CNS glycoprotein epitope with essential roles to play in neural plasticity, higher brain function, synaptic plasticity, spatial learning and memory. However, it is not limited to the CNS and also displays specific localisations in other strategic locations elsewhere in the human body including in the kidney [208], the heart [209,210], retina [211] and as a component of the PNNs identified in the auditory system [212]. As a neural cell marker, HNK-1 plays crucial roles in cell migration and cellular attachment during embryonic nerve development and formation of the notochord from the neural tube (Figure 9). Carbohydrate–protein interactions between HNK-1 reactive sulpho-glucuronyl-glycolipids and PG lectin domains mediate neuronal cell adhesion and neurite outgrowth [213–215]. Some laminin isoforms also bind specifically to sulphated glycolipids [216] and are important in cell adhesion [215], particularly in nerve development. Several neural cell-adhesion molecules contain the HNK-1 epitope including neural cell adhesion molecule (NCAM), myelin associated glycoprotein (MAG), myelin basic protein (MBP), neural-glial adhesion molecule (Ng- CAM, L1), contactin, P0, Tenascin-C, Tenascin-R. Sub-populations of the enzymes *N*-acetylcholinesterase and 5′ -nucleotidease also contain the NHK-1 epitope, which is important in their localisation in synaptic vesicles and membranes. Acetyl cholinesterase and 5′ -nucleotidase are GPI anchored ecto-enzymes of high catalytic efficiency. Acetylcholinesterase cleaves the neurotransmitter acetylcholine in the neuromuscular junction (NMJ) and this allows muscles to return to a relaxed state following contraction. Acetylcholinesterase cleaves in excess of 5000 molecules of acetylcholine/s per molecule of enzyme. Without such an efficient enzymatic system and co-ordinated expression of neurotransmitters in the NMJ, muscles would be tensed and relaxed in an uncoordinated manner and movements would be jerky and irregular as evident in neuromuscular disorders such as Parkinson's disease or the spastic paralysis evident in Schwartz–Jampel Syndrome. It is not surprising therefore that the expression of NHK-1 is under strict spatial and temporal regulation on migrating neural crest cells, cerebellum, myelinating Schwann cells and motor neurons but not on sensory neurons. A form of HNK-1 substituted aggrecan is synthesised in the notochord and in early foetal rudiment cartilage [90,92] (Figure 9a,b). HNK-1 has been immunolocalised to the electro-receptors of the shark and electric organs of the electric eel (*Electrophorus electricus*), electric catfish (*Malapterurus electricus)* and electric ray (*Torpedo marmorata)*. HNK-1 has also been mapped to electroconductive tissue during human foetal heart development and is expressed by cultured cardiomyocytes leading to the development of smart electroconductive

polymers for applications in regenerative medicine [217]. HNK-1 sulphotransferase (HNK-1ST) catalyses the transfer of sulphate to position 3 of terminal glucuronic acid in protein and lipid linked oligosaccharides carried by many neural recognition molecules [218,219]. These facilitate cellular interactions during CNS development and in synaptic plasticity. HNK-1 ST acts in combination with two other glucuronyl transferases (GlcAT-P and GlcAT-S) to form a heteromeric complex in the biosynthesis of the HNK-1 trisaccharide epitope [220,221]. HNK-1 ST suppresses the glycosylation of α-dystroglycan in sub-populations of melanoma cells in a number of tissues where neither GlcAT-P nor GlcAT-S is expressed, and this reduces the ligand binding capability of α-dystroglycan establishing a tumour suppressor role for HNK-1 ST in melanoma. The HNK-1 epitopes of acetylcholinesterase and 5′ -nucleotidase have roles in cell–cell and cell–matrix communication independent of their enzymatic activities.

HNK-1 (SO4-3GlcAβ1-3Galβ1-4GlcNAc) is expressed on *N*-linked and *O*-mannose linked glycans in the nervous system. Several proteoglycans bear the HNK-1 epitope including phosphacan and aggrecan (Figure 3b,c). NHK-1 sulphotransferase can utilise the xylose-galactose-galactose-glucuronic acid linkage tetrasaccharide as acceptor to attach the 3-*O* sulphate group to the non-reducing glucuronic acid residue [222] but in so doing inhibits further CS chain elongation; thus, aggrecan substituted with HNK-1 has a lower density of CS chains and has a reduced hydrodynamic size compared to cartilage aggrecan [220,222]. Phosphacan occurs as a soluble PG and as a variant protein tyrosine phosphatase which contains KS and CS side chains in addition to HNK-1 carbohydrate. The HNK-1 motif in phosphacan is *O*-mannose linked through an Asn on the core protein. Notochordal and early rudiment cartilage cells synthesise a form of aggrecan substituted with the HNK-1 epitope, but this disappears in later stages of skeletal development (Section 9 and Figure 9).

The HNK-1 substituted Tenascin-R and -C splice variant multimeric ECM glycoproteins contain multiple FNIII and EGF repeats and a fibrinogen domain which are interactive with the C type lectin domains of the lectican CS PG family in brain [223,224]. Tenascin-R is a major component of the PNNs which surround neurons in the brain, spinal cord and in specific areas of the hippocampus [225]. Perineuronal nets consist of the lectican CS-PGs assembled into extracellular networks through interaction with HA and link proteins cross-linked by Tenascin-R and are linked to neurons through their C-terminal domains, endowing them with neuroprotective properties [226] (Figure 7a–e,k).
