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Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Weigel PH - Int J Cell Biol (2015)

Bottom Line: Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains.The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes.These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

ABSTRACT
Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

No MeSH data available.


Schematic model of HAS showing the functions needed for HA chain growth at the reducing end and transfer to the cell surface. HAS uses multiple discrete functions (numbers 1–7) to assemble each HA disaccharide (red squares are GlcNAc and green circles are GlcUA). The same HAS protein is indicated in two different situations, at sequential times, as it alternately adds HA-GlcUA-UDP to a new GlcNAc-UDP, using functions 1, 3, and 5 (left), and then adds HA-GlcNAc-UDP to a new GlcUA-UDP, using functions 2, 4, and 6 (right). In this example (variant 1; Table 1) the sugar-UDPs are sequentially added in a continuous alternating manner and each cohort of needed functions cycles between being active (larger black numbers) and inactive (smaller gray numbers) within the active site domains (gray ovals). The functions required to add GlcNAc-UDP to HA-GlcUA-UDP are (left): 1, GlcNAc-UDP acceptor binding; 3, HA-GlcUA-UDP donor binding; 5, HA-GlcUA-UDP: GlcNAc-UDP, β1,3(HA-GlcUA-) transferase; and 7, HA translocation through the membrane. The functions required to add GlcUA-UDP to HA-GlcNAc-UDP are (right): 2, GlcUA-UDP acceptor binding; 4, HA-GlcNAc-UDP donor binding; 6, HA-GlcNAc-UDP: GlcUA-UDP, β1,4(HA-GlcNAc-) transferase; and 7, HA translocation.
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fig1: Schematic model of HAS showing the functions needed for HA chain growth at the reducing end and transfer to the cell surface. HAS uses multiple discrete functions (numbers 1–7) to assemble each HA disaccharide (red squares are GlcNAc and green circles are GlcUA). The same HAS protein is indicated in two different situations, at sequential times, as it alternately adds HA-GlcUA-UDP to a new GlcNAc-UDP, using functions 1, 3, and 5 (left), and then adds HA-GlcNAc-UDP to a new GlcUA-UDP, using functions 2, 4, and 6 (right). In this example (variant 1; Table 1) the sugar-UDPs are sequentially added in a continuous alternating manner and each cohort of needed functions cycles between being active (larger black numbers) and inactive (smaller gray numbers) within the active site domains (gray ovals). The functions required to add GlcNAc-UDP to HA-GlcUA-UDP are (left): 1, GlcNAc-UDP acceptor binding; 3, HA-GlcUA-UDP donor binding; 5, HA-GlcUA-UDP: GlcNAc-UDP, β1,3(HA-GlcUA-) transferase; and 7, HA translocation through the membrane. The functions required to add GlcUA-UDP to HA-GlcNAc-UDP are (right): 2, GlcUA-UDP acceptor binding; 4, HA-GlcNAc-UDP donor binding; 6, HA-GlcNAc-UDP: GlcUA-UDP, β1,4(HA-GlcNAc-) transferase; and 7, HA translocation.

Mentions: Streptococcal and mammalian HASs in membranes [23–26] or as purified enzymes [27] elongate HA at the reducing end and do not require an exogenous primer to begin HA synthesis. HAS initiates biosynthesis using just the two sugar-UDP substrates, although we now know that the enzyme makes a self-primer using only GlcNAc-UDP (described below). HAS is an unusual enzyme in that it uses four substrates (i.e., two sugar-nucleotides and two types of HA-UDP chains, with either GlcUA or GlcNAc at the reducing end) and two glycosyltransferase activities within the same protein. DNA and RNA polymerases utilize template molecules to direct synthesis of products with only one type of bond between monomers. Heteropolysaccharide synthases, such as HAS (which makes a [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP polymer), create de novo two different glycoside linkages in an alternating manner. The HA product after each sugar addition then becomes a substrate for the next sugar addition. In the presence of exogenous precursors, membrane-bound HASs use at least seven binding or catalytic functions (Figure 1) to synthesize disaccharide units at the reducing end of a growing HA-UDP chain. Class I HAS enzymes are processive; they do not rebind and extend HA chains once they are released.


Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Weigel PH - Int J Cell Biol (2015)

Schematic model of HAS showing the functions needed for HA chain growth at the reducing end and transfer to the cell surface. HAS uses multiple discrete functions (numbers 1–7) to assemble each HA disaccharide (red squares are GlcNAc and green circles are GlcUA). The same HAS protein is indicated in two different situations, at sequential times, as it alternately adds HA-GlcUA-UDP to a new GlcNAc-UDP, using functions 1, 3, and 5 (left), and then adds HA-GlcNAc-UDP to a new GlcUA-UDP, using functions 2, 4, and 6 (right). In this example (variant 1; Table 1) the sugar-UDPs are sequentially added in a continuous alternating manner and each cohort of needed functions cycles between being active (larger black numbers) and inactive (smaller gray numbers) within the active site domains (gray ovals). The functions required to add GlcNAc-UDP to HA-GlcUA-UDP are (left): 1, GlcNAc-UDP acceptor binding; 3, HA-GlcUA-UDP donor binding; 5, HA-GlcUA-UDP: GlcNAc-UDP, β1,3(HA-GlcUA-) transferase; and 7, HA translocation through the membrane. The functions required to add GlcUA-UDP to HA-GlcNAc-UDP are (right): 2, GlcUA-UDP acceptor binding; 4, HA-GlcNAc-UDP donor binding; 6, HA-GlcNAc-UDP: GlcUA-UDP, β1,4(HA-GlcNAc-) transferase; and 7, HA translocation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Schematic model of HAS showing the functions needed for HA chain growth at the reducing end and transfer to the cell surface. HAS uses multiple discrete functions (numbers 1–7) to assemble each HA disaccharide (red squares are GlcNAc and green circles are GlcUA). The same HAS protein is indicated in two different situations, at sequential times, as it alternately adds HA-GlcUA-UDP to a new GlcNAc-UDP, using functions 1, 3, and 5 (left), and then adds HA-GlcNAc-UDP to a new GlcUA-UDP, using functions 2, 4, and 6 (right). In this example (variant 1; Table 1) the sugar-UDPs are sequentially added in a continuous alternating manner and each cohort of needed functions cycles between being active (larger black numbers) and inactive (smaller gray numbers) within the active site domains (gray ovals). The functions required to add GlcNAc-UDP to HA-GlcUA-UDP are (left): 1, GlcNAc-UDP acceptor binding; 3, HA-GlcUA-UDP donor binding; 5, HA-GlcUA-UDP: GlcNAc-UDP, β1,3(HA-GlcUA-) transferase; and 7, HA translocation through the membrane. The functions required to add GlcUA-UDP to HA-GlcNAc-UDP are (right): 2, GlcUA-UDP acceptor binding; 4, HA-GlcNAc-UDP donor binding; 6, HA-GlcNAc-UDP: GlcUA-UDP, β1,4(HA-GlcNAc-) transferase; and 7, HA translocation.
Mentions: Streptococcal and mammalian HASs in membranes [23–26] or as purified enzymes [27] elongate HA at the reducing end and do not require an exogenous primer to begin HA synthesis. HAS initiates biosynthesis using just the two sugar-UDP substrates, although we now know that the enzyme makes a self-primer using only GlcNAc-UDP (described below). HAS is an unusual enzyme in that it uses four substrates (i.e., two sugar-nucleotides and two types of HA-UDP chains, with either GlcUA or GlcNAc at the reducing end) and two glycosyltransferase activities within the same protein. DNA and RNA polymerases utilize template molecules to direct synthesis of products with only one type of bond between monomers. Heteropolysaccharide synthases, such as HAS (which makes a [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP polymer), create de novo two different glycoside linkages in an alternating manner. The HA product after each sugar addition then becomes a substrate for the next sugar addition. In the presence of exogenous precursors, membrane-bound HASs use at least seven binding or catalytic functions (Figure 1) to synthesize disaccharide units at the reducing end of a growing HA-UDP chain. Class I HAS enzymes are processive; they do not rebind and extend HA chains once they are released.

Bottom Line: Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains.The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes.These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

ABSTRACT
Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

No MeSH data available.