The structure of secreted mucins isolated from the adherent mucus gel: comparison with the gene products.

In the colon there is acontinuous, insoluble adherent mucus gel covering the surface mucosa and forming a protective barrier. The gel forming properties of the mucus barrier are dependent on the component mucins. Mucin subunits consisting of a central protein core with glycosylated and non-glycosylated regions are joined together by disulphide bridges (between the non-glycosylated regions) to form polymers.(~)Following exhaustive proteolysis only the glycosylated region of the subunit remains intact. The polymeric structure of mucin is essential for it's gel forming and viscous properties.@) At present 7 mucin genes have been partially characterised, 6 of which are secreted mucins (MUC 2-7).(3.4)Sequencing has essentially been confined to the major glycosylated region of the mucin core which consists of tandemly repeated amino acid sequences. For each MUC gene the number of tandem repeats varies as does the number andcomposition of aminoacidscharacteristic ofthese tandem repeats. Although all the tandem repeats are rich in threonine and to a lesser extentserine, theproportionofthreoninetoserinevaries. MUC 3,4,5,6 & 7 have thr:ser ratios of 1.4: I , 1.3: 1,2: I , 1.8: 1 & 1.4: 1 respectively, , whereas serine is absent from MUC2. At least three of these genes, MUC 2,3 &4, have been shown tobeexpressedin thecolon. MUC 2 (which is expressed in the colon) is the best characterised mucin gene, it's sequence being essentially complete apart from the amino terminal.(5*6)It comprises -4700 aa and has two glycosylated repetitive regions of different molecular size; a23 aa tandem repeat region of -23OOaa which is very rich in thr, 60% of the amino acids, and a347 aa base pair repeat region containing 49% thr and 10% ser. Cys rich regions are located both at the carboxyl and amino ends of the molecule. This study set out to investigate the structure of human and pig colonic mucin isolated from the secreted adherent mucus gel and to reconcile these structures with the identified geneproducts. Glycoprotein from pig and human colonic mucus was extracted in proteinase inhibitors and purified by equilibrium centrifugation in 3.5M CsCl followed by gel filtration on Sepharose CL-2B. Reduced glycoprotein was produced by reduction with mercaptoethanol, 24 hr, 20oC; proteolytically digested mucin by digestion with papain 0 . 0 8 ~ mg-lmucin, 60OC, 48 hr.The molecular weight of mucin was determined by sedimentation equilibrium, using a Beckman Model E analytical ultracentrifuge, and multi-angle light scattering. Dilute solution viscosity measurements were made using aCouette rotating vis~ometerat25~C.Thiol analysis was by the methodofMantleetaf. ( I 990)(6) Purified mucin was reduced with 0.33M NaBH,, thiol groupslabelledwith4,4dithyopyridine.The number oftotal and free thiols were calculated using the molar absorption coefficient. Amino ac id a n a l y s i s w a s by the me thod of Car l ton and Morgan ( 1 988).(7)Samples hydrolysedfor24 hr at 1 10°C were derivatised with 9 fluorenylmethylchloroformate and analysed by reverse phase HPLC. Purified mucin glycoproteins were largely excluded (60%) from Sepharose CL-2B. Reduction of mucin produced a broad single peak which was also mainly excluded. Digestion produced a smaller species includedon SepharoseCL-2B (Kav.0.56). The weight average molecular weight of polymeric and digested mucin was determined by two methods, sedimentation equilibrium and light scattering. Measurements from both methods were in close agreement. The M,forpolymeric and digested mucin weredetermined to be5.7x1O6and5.5xlO5respectively. The papaindigested mucin was asingle sizedpolydisperse species. The weight average molecular weight of reduced mucin determined by light scattering was 3 . 3 ~ 106. This size distribution was confirmed by intrinsic viscosity studies. Polymeric, reduced and digested mucin having intrinsic viscosity values of0.27,O. 10 and0.02 mg ml-'respectively . Polymeric colonic mucincontained 35 23.1 nmol mg-1 total thiols including 7.0 nmol mg-'free thiol groups. Reduced much contained 23i4Snmolmg-1 total thiolsincluding 17nmolmg-1 free thiolgroups. The majority of thiols in polymeric colonic mucin -28 nmol mg-lare involved in disulphide bridge formation. This together with the large number present andthedecreasein molecular sizeon reductionof the isolated mucin is consistent with their involvement in polymerisation. The presence of -3 nmol mg-I disulphide bridges after reduction suggests the presence of intra-molecular disulphide bridges possibly involved in a globular protein structure and presumeably inacessible to the reducing agent. Proteolysis removed all thiols suggesting that they are confined to the non-glycosylated region of the protein core acessible to the proteolytic enzyme. Both human and pig colonic mucin contained a high proportion of ser, thrandpro,atotalof39% and40% by weightoftotalproteincontent respectively. Digestion of pig colonic mucin resulted in loss of -30% of protein by weight from 17% to 12% of the total molecule. Thr and ser were conserved which is characteristic of the glycosylated regions of other mucins.(')The ratio of thr:ser in papain digested humanandpigmucin was-1 : 1. From these results pig colonic much isolated in the presence of proteolytic inhibitors has amolecularweightof5.7~ 106andcontains -14 nmol mg-I disulphide bridges (ie 84disulphide bridgesperpolymer). Eachpolymerconsistsof at least 2 subunits Mr3.3x106linked by disulphide bridges. Each subunit contains 4-6 similar size regions of Mr 5 . 5 ~ 105. The glycosylated region of both human and pig mucin is rich in thr, ser and pro with a thr:ser ratio of -1 : 1. Proteolysis removes all the non-glycosylated part of the mucin to leave the heavily glycosylated protected protein core which presumeably represents the tandem repeat segment of the mucin. Therefore i t is interesting to compare the amino acid analysis of the digested mucins with the published sequences of tandem repeats of the MUC genes. The amino acid analysis for both human and pig glycosylated region has a much higher ser content relative to thr than would be expected for the tandem repeat region ofMUC 2&3, expressed in the human colon. The thr:ser ratio is most compatible with that of the tandem repeat region of MUC4 which has a thr:ser ratio 1.2: I , however the total amino acid composistion of humandigestedmucin isdifferenttothatoftttthe tandem repeat region of MUC4. Further the size of theglycosylated region and the presence of asingle species does notequate with MUC 2. This analysis described here for the secreted colonic mucin is not compatible with the products of the m u c h genes so far sequenced being the major component of the secreted adherent colonic mucus gel.