Biosynthesis of Heparin

Microsomal fraction from mouse mastocytoma was incubated for 60 min with UDP-[Wlglucuronic acid and unlabeled UDP-N-acetylglucosamine, producing nonsulfated labeled polysaccharide. Continued incubation for an additional 60-min period, in the presence of 3’-phosphoadenylylsulfate, yielded sulfated, heparin-like polymer. The incorporation of W was terminated at the beginning of the sulfation period, by including an excess of unlabeled UDPglucuronic acid in the incubation mixtures. The distribution in the nonsulfated and chase-sulfated W-polysaccharides, respectively, of N-unsubstituted, Nacetylated, and N-sulfated glucosamine residues was investigated by selective deamination with nitrous acid. Samples were treated at room temperature with 0.24 M NaNOZ in 1.8 M acetic acid for 80 min (Reaction A) or with 3.9 M NaNOn in 0.28 M acetic acid for 10 min (Reaction B). In Reaction A the glycosidic bonds of glucosamine residues having either unsubstituted or sulfated amino groups were cleaved, whereas in Reaction B N-unsubstituted glucosamine units only were attacked. The effect of deamination on the labeled polysaccharide preparations was studied by gel chromatography of the products, on Sephadex G-25. Characterization of the nonsulfated polysaccharide, by use of these methods, indicated that about half of the glucosamine residues had unsubstituted amino groups. This result was confirmed by ion exchange chromatography, on DEAE-cellulose, which showed the presence of a polymer with less polyanion character than standard hyaluronic acid. After N-acetylation, the elution pattern of this intermediary polysaccharide was the same as that of hyaluronic acid. The sulfated polysaccharide contained N-sulfated and N-acetylated but no N-unsubstituted glucosamine units. It is concluded that N-deacetylation of the heparin precursor polysaccharide occurs in the absence of 3’-phosphoadenylylsulfate and that the amino groups thus exposed can serve as acceptors of sulfate residues. The biosynthrsih of sulfated Rl~cosalnirloglycalls has been studied cstc,nsivrly, and is now fairly well characterized with regard to the principal reactions involved (2, 3). It is generall) agreed that the 1)olysaccharide chain is formed by stepa-ise and alternate t,r:tnsfer of uranic arid and TV-acetylhesosamitle monosaccharitlc units from the appropriate nuclcotide sugars to the nonreducing termini of growing chains. Sulfation occurs subsequent to 1”)lylllc’rizatiot~ and requires the presence of 3’.phosphoadenylylsulfatc. The biosynthesis of Irrparin and heparan sulfate prcscnts a number of spcrific problems relating to t,he unique structural features of these polysaccharides (see review b,v Rod& (2)). The formation of the carbohydrate skeleton of heparin has been thoroughly ill\-cstigatcd by Silbert (4-6), who demonstrated that a microsomal preparation from mast ccl1 tumor of the l>unnPotter type catalyzes the transfer of glucuronic acid and N-acet,ylglucosuminc from the corresponding Til)l’?lcrivatives to an endogclrous acceptor. The resulting product n-as nonsulfated or lowsulfatctl mtl rcscmhled hyaluronic arid in its macromolecular a11(1 1~olyclectrolytc properties; yet it bohavecl like heparin on trcatmcnt with various dcgradativc enzymes (4). By use of N-:~c~etyl-3JI-labclc~~ T:L)P~h’-3cetylg:lucosamirle along with 14Clabcl~~d nuclcotide sugars, the product was shown to contain uranic acid, glucosamine, and Xacctgl in approsimatcly equimolnr proportions (4, 6). In the prcsc~llrc~ of 3’.1)hosphoatlell!-lvlsrrliate a sulfatctl pal\-saccharidP was I’ormcd, iI1 which 40 to 50C;7, of the total sulfatr appeared to 1~ A-sulfate (6). A corresponding loss of A-ncetyl was tlcmonstratcd; this deacctylatioll occurred both when 3’. phosphoadenylylsulfatt: n-as present during ~l?cosarllinoglycali polymerization or when the nucleotide sulfate was added subsequent to the polymerization 1xocess. It \\-as concluded that N-de:tcrtylatioJl orcurs in conjunction with the formation of Nsulfate groups. In addition to t~ntlogenous microsomal polysnccl~aritlc, various exogenous prcpnrations have been used as sulfate acceptors in cell-free cspcrimrnts.. hn enz\-me isolated from Furth mastocytoma by l(:~l:isubr:~Jn:rniaii et al. (7) w-as thus found to catalyze the transfer of sulfate to a number of pol~sarch:lridcs, preferentially into sulfamino groups. N-Desulfatrd hcparin and heparan sulfate \vcre bct,tcxr sulfatr acceptors than n-erc the corresponding intact 1)ol~s:~~cliaritlc,s; in addition, acetylntion of the IV-tlrsulfoheparin rctluccscl tlir sulfate incorporation to less than 20 7; of the iuitinl 1~~1. This ohticrvntion cannot readily br reconciled with a concc~rtc~tl :rc~c~tyl-hulfatr rxchange m(&:ulism, but rathrr