Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
29
pubmed:dateCreated
1996-9-12
pubmed:abstractText
The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits. beta-Hexosaminidase A (alphabeta) is a heterodimer, whereas hexosaminidase B (betabeta) and S (alphaalpha) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only beta-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit. Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta-subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of beta-hexosaminidase A.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
271
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
17377-82
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:8663217-Amino Acid Sequence, pubmed-meshheading:8663217-Base Sequence, pubmed-meshheading:8663217-Carbohydrate Sequence, pubmed-meshheading:8663217-Cloning, Molecular, pubmed-meshheading:8663217-DNA Primers, pubmed-meshheading:8663217-G(M2) Ganglioside, pubmed-meshheading:8663217-Genetic Variation, pubmed-meshheading:8663217-HeLa Cells, pubmed-meshheading:8663217-Hexosaminidase B, pubmed-meshheading:8663217-Humans, pubmed-meshheading:8663217-Isoenzymes, pubmed-meshheading:8663217-Kinetics, pubmed-meshheading:8663217-Macromolecular Substances, pubmed-meshheading:8663217-Molecular Sequence Data, pubmed-meshheading:8663217-Mutagenesis, Site-Directed, pubmed-meshheading:8663217-Recombinant Fusion Proteins, pubmed-meshheading:8663217-Restriction Mapping, pubmed-meshheading:8663217-Substrate Specificity, pubmed-meshheading:8663217-Transfection, pubmed-meshheading:8663217-beta-N-Acetylhexosaminidases
pubmed:year
1996
pubmed:articleTitle
Identification of domains in human beta-hexosaminidase that determine substrate specificity.
pubmed:affiliation
Section on Biochemical Genetics, Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't