Purchase this article with an account.
SV Andersson, C Magnusson, EC Sjögren, S Tågerud, JP Gierow; Sequencing and Expression of the Beta-hexosaminidase Alpha-subunit in Rabbit Lacrimal Gland . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3133.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The lysosomal ß-hexosaminidases occurs as three isozymes, formed by the combination of α- and ß- subunits. ß-hexosaminidase has previously been shown to be present in and secreted from lacrimal gland acinar cells. The purpose was to further characterize the enzyme by determining the full-length HexA mRNA sequence coding for the α-polypeptide and study its expression in tissue and cultured cells from rabbit lacrimal gland. Methods: Total RNA was extracted from rabbit lacrimal gland tissue, unstimulated and stimulated cultured acinar cells. Using the rapid amplification of cDNA ends (RACE) technique, HexA cDNA was produced from tissue RNA. Oligonucleotide primers were designed from conserved regions within human and mouse HexA mRNA sequences. PCR amplified fragments of the correct size were cloned with the pGEM-T vector system and sequenced. For expression studies a PCR amplified fragment (394 bp) was cloned and sequenced. The transcribed 32P-labeled antisense RNA probe was used in studies of HexA mRNA expression in tissue and in cultured acinar cells incubated in the presence or absence of 0.1 mM carbachol and 0.1 mM VIP for 1h. Results: A rabbit HexA cDNA of 1,640 nucleotides, showing 81% and 78% identity with human and mouse sequences, 2255 bp resp. 1960 bp in length, has been determined. It includes a suggested open reading frame coding for 436 amino acids. The translated peptide exhibited 90% and 86% identity with the human (529 aa) and mouse (528 aa) HexA proteins. Northern blot analyses showed an increased HexA mRNA expression in unstimulated and stimulated cultured lacrimal gland acinar cells compared to lacrimal gland tissue . Conclusion: The HexA gene is strongly conserved within mammals, suggesting that the rabbit sequence should be of similar length. Further 5’ end sequence analysis is necessary to obtain the region containing the transcription start codon. The enhanced HexA mRNA expression in cultured cells could be a result of the purification of the acinar cells during cell isolation, or could suggests a role, yet unknown, for HexA in the formation and stabilization of acinar structures, e.g. by modulation of the surrounding extracellular matrix.
This PDF is available to Subscribers Only