Sequencing, expression, and enzymatic characterization of beta-hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells.
The lysosomal enzyme, beta-hexosaminidase, exists as two major isoforms; HexA and HexB. HexA is an alpha beta-subunit heterodimer and HexB a beta-subunit homodimer. Both isoforms can remove nonreducing beta-N-acetyl-D-glucosamine residues, whereas HexA hydrolyzes charged substrates as G(M2) gangliosides as well. beta-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 alpha- and beta-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 beta-hexosaminidase antibodies are unable to recognize the rabbit enzyme, cloning and sequencing of the alpha- and beta-subunits were performed. Sequencing of the these subunits indicate that both are highly conserved between human, mouse, and rabbit. In contrast to the beta-subunit, showing an even mRNA expression between tissue and cultured cells, the level of alpha-subunit expression was higher in cultured acinar cells compared to tissue, with no alteration after cell stimulation. A minor but significant increase in total beta-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 beta-hexosaminidase in lacrimal gland and cultured acinar cells.
Andersson
Sofia V
SV
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE- 391 82 Kalmar, Sweden.
Sjögren
Elsie C
EC
Magnusson
Caroline
C
Gierow
J Peter
JP
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000166:cytology* / Q000201:enzymology*; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D016415:Sequence Alignment; D015854:Up-Regulation / Q000235:genetics; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D021122:Protein Subunits; D012333:RNA, Messenger; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases