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Preparation of Conjugates of Proteins with Amyloses by Elongation Of BrooRGANtccHEMrsrRv 21, 319-329 ( 1993) Preparationof Conjugatesof Proteinswith Amyloses by Elongationof CovalentlyAttached Primers Using Glycogen Phosphorylasea1 YeN-Ho CHu nNo GeoncE M. WHrrr,sroesr Depurtment rf'Chemisnl:, Huruurd Uniuersitt', l2 O.r.fltrdStreel Cumbridpe, Mussuchusetts02 I 38 ReceiuedJunuurt' I l, 199-1 This paperdescribes convenient preparations of protein-amyloseconjugates. These prepa- rations are based on the elongation of maltooligosaccharidesby reaction with glucose-l- phosphate, catalyzed by glycogen phosphorylaserr. Coupling of maltooligosaccharidesto proteins by reductive amination generated covalently attached primers tbr the glycogen phosphorylaserr-catalyzed polymerization of glucose-l-phosphate. SDS-polyacrylamide gel tlP electrophoresiswas usefulfor characterizingthese conjugates. NMR \pectr()\copy could be used conveniently to assay simultaneouslythe formation and the enzymatic activity of the amylose-conjugatedproteins. This assay could be used directly in reactions catalyzed by glycogen phosphorylaserr; it should also be applicable to protein mixturer of greater complexity than the ones used here. Methods of synthesizingenzymatically active neoglyco- proteins having a range of molecular weights are described. r 1993Academic Prcss. lnc INTRODUCTION The objective of this work was to develop methods to prepare conjugates of proteinswith high molecularweight amyloses.We wishedto comparethe proper- tiesof this classof neoglycoproteinswith thoseof naturallyoccurring glycoproteins and with conjugatesof proteinswith poly(ethyleneglycol) (PEG) and other man- madepolymers. The oligosaccharidemoieties of glycoproteinsmay play a number of roles: maintainingprotein conformationand solubility (1); stabilizingthe poly- peptide against proteolysis (2); functioning in processing,intracellular sorting, and excretion of glycoproteins(J); mediatingbiological activity (4): and serving as cell-surfacelabels in differentiationand development (1,5). Functions de- pending on biological recognition of sugar groups undoubtedly require specific oligosaccharidestructure; functions depending on more general physical proper- ties (solubility, thermal stability, resistance to proteases, and masking against recognition by antibodies) may be replicable using simpler oligosaccharidemoie- I This work was supported by the National Institute of Health (GM30367) and by the National ScienceFoundation under the EngineeringResearch Initiative to the M.LT. BiotechnologyProcessing Engineering Center (Cooperative Agreement CDR-88-03014). 2 To whom corresnondenceshould be addressed. 319 004_s-2068/93$-5.00 Copyright O 1993 by Academic Press. Inc. All rights of reproduction in any firrm reserved. 320 CHU AND WHITESIDES ties. Conjugatesof proteins with PEG have shown a number of interestingproper- ties and are now being introduced into clinical practice on the basis, inter alia, of low immunogenicity and long serum lifetime (6). Direct attachment of preformed polysaccharidesto proteins is difficult for sev- eral reasons.It is difficult to functionalize a polysaccharideselectively in a particu- lar position (e.g., a terminal position), difficult to achieve reaction of many poly- mers with surfacesor high molecular weight reactantsonce activated, and difficult to characterize the products. We have taken a different approach (Eq.[]). We phosphate buffer -tr Hor bsoru,pHzo Hol 'NFt3-(cH2)4-cHl.. --> rrl tcrc),ffi!$-o* + ------- (crc)'fff$$-,NH2 L'l NaBH3cN .oH (cH2)a-i* a\*, rd modify the protein of interest by attachment of a low-molecular-weightoligosac- charide, derived from maltohexose (penta[a-o-glucopyranosyl-(I - 4)]-o-gluco- pyranose)or its derivatives. The resulting protein-maltohexoseconjugates can be characterized,at leastto the extent of establishingthe number. even if not the specific location, of attached oligosaccharides.These covalently attached malto- hexosederivatives act as primers for the polymerizationof glucose-l-phosphate (Glc-l-P) catalyzedby phosphorylasen (seebelow). This system has the advantagesthat the protein-maltohexose conjugates can be characterized and that the in silrupolymerization catalyzedby phosphorylase a is compatiblewith retention of activity in the protein of interest.The amylose chain attached to the protein can also be shortened with retention of activity by treatment with phosphorylasea and phosphatein the absenceof Glc-l-P or by hydrolysiswith amylase(1,4-a-o-glucan glucanohydrolase, EC 3.2.1.1).Charac- terizingthe protein-amyloseconjugates-especially the degreeof polymerization of the amylosechains and the numberof primersparticipating in the polymerization reactions-remains a problem that we have only partially solved. The production of linear and branched amyloseshas been developed exten- sively, especially by Ziegast and Pfannemriller(7). We have described the initial results of our work in an earlier paper (B). We selectedcytochrome ('(from horse heart) (9), ribonucleaseA (RNase A, EC 3.1.21.5,from bovine pancreas)(10), and carbonic anhydrase (CA, carbonate hydrolyase, EC 4.2.1.1. from bovine erythrocytes) (11) as model proteins for several reasons. All are commercially available,inexpensive, and well characterized.CA is monomeric,with a molecular weight of 30 kDa. It contains 19 lysine residues, all of which are on the surface of the protein and accessibleto the modifying agents. Assaying the catalytic activity of CA is straightforward(11). RNase A is a single polypeptide, with a molecular weight of l4 kDa and has l0 lysine residues.One of these (Lys-41) is at the active site of the enzyme (10). We have developeda simplifiedassay system for RNase A.3 Cytochrome c' is also monomeric, with a molecular weight of 12.4 I The assayfor activity of RNase using rlP NMR spectroscopywas developedin our laboratory by Rajeeva Singh. PROTEIN-AMYLOSECONJ UGATES 321 kDa. It possessesl8 lysines,all on the surfaceof the protein. Cytochrome c' has a strong absorption at 409 nm (eosn/e280: l7) and is easily assayedspectrophoto- metrically (9). Glycogenphosphorylase (.1 ,4-a-o-glucan:orthophosphate a-o-glucosyltransfer- ase,EC 2.4.1.1)catalyzes the reversiblepolymerization of Glc-l-P (Eq. [2]).The best-studiedisozyme of phosphorylaseis that from K=3.6 a-o-Glc-l-P+ ta(1,4)-GlcJ,,<- fa(1,4)-GlcJ,t P; I2l rabbit skeletal muscle (12).0The enzyme is highly specific with respect to its substrates.Only a-o-Glc-l-P reacts in the transfer of a glucosyl residue. This enzyme requiresa primer as the secondsubstrate to effectpolymerization. Malto- tetraose (i.e., trila-o-glucopyranosyl-(l-+ 4)-o-glucopyranose)is the minimum oligomerthat will function as primer. The equilibriumconstant for the polymeriza- tion reaction (Eq. [2]) at neutral pH is 3.6 and favors glycogensynthesis in uitro (1, 1J).5 EXPERIMENTAL PROCEDURES Materials Proteins and enzymes used were obtainedfrom Sigma (St. Louis, MO): cyto- chrome r' (from horse heart), ribonucleaseA (from bovine pancreas).carbonic anhydrase(from bovine erythrocytes),sucrose phosphorylase (from Lerrcortostoc' mesenteroides),and glycogen phosphorylasen. Maltohexoseand maltoheptose (i.e., hexafa-o-glucopyranosyl-(l--- 4)]-o-glucopyranose)were purchasedfrom Boehringer-MannheimBiochemicals (Indianapolis. IN). Maltose (i.e., a-o-gluco- pyranosyl-(| -+ 4)-o-glucopyranose),sodium borocyanohydride, and sucrosewere commercialproducts of Aldrich (Milwaukee,Wl).2' ,3'-cCMP,3'-CMP, glucose- l-phosphate,poly(A), and p-nitrophenyl acetatewere obtainedfrom Sigma. Re- agentsand apparatus(e.g., mini PROTEAN II) usedfor polyacrylamidegel electro- phoresis (PAGE) were products of Bio-Rad (Richmond, CA). SDS-PAGE was carried out following the manufacturer's procedure. I Themostactiveformofrabbitmuscleglycogenphosphorylaseisadimeroftwoidenticalmonomers. w'ith tt42 amino acid residues and a molecular weight of 97,440 Da fbr each subunit. This enzyme contains a pyridoxal phosphate :rs a cofactor. covalently bound via a Schiff base to an active site lysine (Lys-6t10).In the restingstate. the enzyme existsas the inactivephosphorylase b, which may be activated by AMP. In responseto nervous or hormonal stimulation, the b form is phosphorylated and becomesthe rr forml phosphorylasea is no longerdependent on AMP for activity. The interconver- sion of phosphorylasea and phosphorylaseb involves the phosphorylationof a single serine residue (Ser-l4) by phosphorylaseb kinaseand its dephosphorylationby the enzyme phosphorylaserr phospha- tase(12). 5 Although the polymerizationreaction is favored at neutral pH in uitro, the reactionin uiuo proceeds toward the degradationof glycogen becausethe intracellularconcentration of P; greatly exceedsthat of Glc-l-P and becauseGlc-l-P is rapidly convertedto Glc-6-Pby phosphoglucomutase(1J). 322 CHU AND WHITESIDES Preparation of C1'toc'hromec:-Maltoheptose Coniugates Conjugation was carried out using a modificationof the published procedure (14). In a typical experiment, lyophilized cytochrome ('(trom horse heart) was addedto a phosphatebuffer (250mrvl, pH 7.0).To this protein solutionmaltohep- toseand sodiumcyanoborohydride, each dissolved in the samebuffer, were added to final concentrationsof l0 mg/ml for the protein and 200and 8l mg/ml. respec- tively, for maltoheptoseand sodium cyanoborohydride.Reaction was carriedout at ambient temperature,the reaction mixture was sampled at various reaction times, and the sampleswere dialyzed and lyophilized.The contentsof carbohy- drate in these conjugateswere determinedby the anthrone-sulfuricacid method (15).Concentrations
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