Sequencing, expression, and enzymatic characterization of β-hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells

The lysosomal enzyme, β-hexosaminidase, exists as two major isoforms; HexA and HexB. HexA is an αβ-subunit heterodimer and HexB a β-subunit homodimer. Both isoforms can remove nonreducing β-N-acetyl-D-glucosamine residues, whereas HexA hydrolyzes charged substrates as GM2 gangliosides as well. β-Hexosaminidase is present in both human and rabbit tear fluid and is secreted from rabbit lacrimal gland acinar cells in primary culture on stimulation with secretagogs. To further characterize the enzyme, the α- and β-subunit mRNA expression was explored in rabbit lacrimal gland tissue as well as in cultured cells. Possible correlation between mRNA expression and HexA specific enzymatic activity was also investigated. Because existing β-hexosaminidase antibodies are unable to recognize the rabbit enzyme, cloning and sequencing of the α- and β-subunits were performed. Sequencing of the these subunits indicate that both are highly conserved between human, mouse, and rabbit. In contrast to the β-subunit, showing an even mRNA expression between tissue and cultured cells, the level of α-subunit expression was higher in cultured acinar cells compared to tissue, with no alteration after cell stimulation. A minor but significant increase in total β-hexosaminidase as well as HexA activity was observed in cultured cells compared to tissue. Enzymatic activity assays also revealed that HexA is the dominating isoform of β-hexosaminidase in lacrimal gland and cultured acinar cells.