Pennybacker M, Liessem B, Moczall H, Tifft C J, Sandhoff K, Proia R L
Section on Biochemical Genetics, Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Jul 19;271(29):17377-82. doi: 10.1074/jbc.271.29.17377.
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.
溶酶体β-己糖胺酶是由α和β亚基组成的二聚体。β-己糖胺酶A（αβ）是异二聚体，而己糖胺酶B（ββ）和S（αα）是同二聚体。尽管各亚基含有高度的氨基酸同源性，但每个亚基都表达一个独特的活性位点，该活性位点可通过水解带电荷底物的不同能力来区分。β亚基上的位点主要降解中性底物，而α亚基位点除此之外还对硫酸化底物有活性。对于糖脂底物也表现出同工酶特异性。在人类同工酶中，只有β-己糖胺酶A与GM2激活蛋白一起才能以具有生理意义的速率降解天然底物GM2神经节苷脂。为了鉴定决定底物特异性的人类β-己糖胺酶亚基的结构域，我们构建了含有α和β亚基序列的嵌合亚基。嵌合构建体在HeLa细胞中表达以筛选活性，然后在杆状病毒表达系统中产生选定的构建体，以评估它们在GM2激活蛋白存在下降解GM2神经节苷脂的能力。产生针对硫酸化底物的活性需要将两个不连续的α亚基序列（氨基酸1-191和403-529）替换到β亚基的相应位置。仅包含这些区域之一并与β亚基序列相连的嵌合构建体要么仅显示中性底物活性（氨基酸1-191），要么完全缺乏酶活性（氨基酸403-529）。当单独表达时，嵌合体和野生型亚基均未显示出激活剂依赖性的GM2水解活性。然而，一个包含与β氨基酸225至556融合的α氨基酸1-191的嵌合亚基，当与野生型α亚基共表达时，显示出与由α和β亚基共表达形成的重组β-己糖胺酶A相当的活性。该结果表明，β亚基氨基酸225-556在β-己糖胺酶A的GM2水解活性中起重要作用。
