Abstract
The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.
Highlights
The relationship between maturation of lipoprotein Lipoprotein lipase (LPL)’ is an asparagine-linked glycoprolipase(LPL)anditstranslocationfromthe endo- tein that is synthesized in a variety of extrahepatic tissues, plasmic reticulum (ER) to the Golgi complex was de- including adipose tissue, heart, skeletal muscle, brain,and termined by measuringlipolytic activity under condi- ovary.Following its synthesis by the parenchyma of these tions preventing transporot f the enzyme from theER tissues, LPL is secreted and bound to heparan sulfate proteoto theGolgi compartment
The monplex, pro-6 Chinese hamster ovary cells accumulated oglycerides andfatty acids liberated are utilized by catalytically active LPL, while secretioofnthe enzyme subjacent tissues for storage or oxidation [1,2,3]
LPL retained intracellularly The response of LPL tonutritional or hormonal changes is bybrefeldin A treatment possessed oligosaccharide a tissue-specific event that is often regulated at a posttranschains that wereprocessed to thecomplex form by the lational level [4,5,6].This regulation may require modifications
Summary
LPL retained intracellularly The response of LPL tonutritional or hormonal changes is bybrefeldin A treatment possessed oligosaccharide a tissue-specific event that is often regulated at a posttranschains that wereprocessed to thecomplex form by the lational level [4,5,6].This regulation may require modifications. Among the modifications known to occur the retained enzyme again remained catalytically ac- posttranslationally, asparagine-linked glycosylation [7] and tive thoeligosaccharides remainedin the highdimerization [8, 9] have been suggested to be interrelated mannose form. The role of an additional modification, boxyl terminusof LPL resulted in intracellularly sulfation of one of the LPL oligosaccharide chains [12], is retained enzyme that wasfully active
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