Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Abstract

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.

Importance of Glycosylation on Function of a Potassium Channel in Neuroblastoma Cells

“…Wild type Kv3.1 migrated as a doublet with a predominant slower migrating band (≈132 kDa) and a very faint faster migrating band (≈88 kDa) (Figure 1A). As previously shown, the upper and lower bands represent attachment of two complex and simple N -glycans, respectively, to the Kv3.1 protein [11], [27]. When both sites were abolished (N220Q/N229Q), a single migrating species (≈81 kDa) was detected which corresponded to the unglycosylated Kv3.1 protein [5].…”

“…Heterologous expression of wild type, N220Q, N229Q, and N220Q/N229Q Kv3.1 proteins in B35 cells were utilized to generate Kv3.1 proteins with and without N -glycans. Previously, we have shown that sialylated N -glycans associated with the Kv3.1 glycoprotein in transfected B35 cells and adult rat brain [11]. Immunoband shift assays of wild type and mutant Kv3.1 proteins revealed that both sites of the Kv3.1 protein expressed in B35 cells could be occupied by sialylated N -glycans.…”

“…Recently, metabolic labeling studies showed that the Kv3.1 channel heterologously expressed in B35 cells generates the Kv3.1 glycoprotein with sialylated N -glycans [11]. Here we have utilized this same expression system to ascertain whether N -glycans associated with the Kv3.1 glycoprotein could influence conducting and non-conducting properties of the Kv3.1 channel.…”

“…Here we have utilized this same expression system to ascertain whether N -glycans associated with the Kv3.1 glycoprotein could influence conducting and non-conducting properties of the Kv3.1 channel. Wild type Kv3.1 protein has two N -glycosylation sites in the first extracellular loop which are occupied in native tissue and heterologous expression systems [4], [5], [10], [11]. Kv3.1 N -glycosylation mutants were constructed by highly conserved mutations of the Asn residues to Gln residues (N220Q/N229Q, N220Q and N229Q) [5].…”

“…Recently, it was demonstrated that the Kv3.1, 3.3, and 3.4, and Kv1.1, 1.2, and 1.4 proteins of adult rat brain, as well as Kv3.1 heterologously expressed in B35 cells, contain sialylated N -glycans [11]. To date, studies of N -glycosylation processing on Kv channels have been conducted utilizing non-excitable cell systems.…”