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Purification and binding properties of a human ficolin-like protein
Journal of Immunological Methods 204 Ž1997. 43–49
Purification and binding properties of a human ficolin-like protein Y. Le a , S.M. Tan a , S.H. Lee b, O.L. Kon a , J. Lu
a,)
a
b
Department of Biochemistry, Faculty of Medicine, National UniÕersity of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Department of Pathology, Faculty of Medicine, National UniÕersity of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 26 November 1996; revised 14 January 1997; accepted 10 February 1997
Abstract Ficolin was initially identified from porcine uterus as a TGF-b 1 binding protein and is considered to have an overall structure similar to that of the complement protein C1q and the collectins. Recent studies have shown that human ficolin is synthesized mainly by monocytes in peripheral blood and that it could potentially bind to sugar structures on microorganisms. The aim of the present investigations was to isolate ficolin from human plasma by affinity chromatography on immobilized sugars. A human serum protein was identified in the GlcNAc eluate from GlcNAc-Sepharose which migrated as a polypeptide of approx. 40 kDa on SDS-PAGE under reducing conditions and was, after further purification by FPLC on a mono-Q column, shown to have an identical N-terminal sequence, over the first 14 residues, to P35, a plasma protein having similar sequence and domain organisation to ficolin. This protein, named the ficolin-like protein, was shown to be sensitive to collagenase and similar to P35 in that it was also disulphide-linked into an oligomer of approx. 320 kDa. However, unlike P35, its binding to GlcNAc was independent of Ca2q. Gel-filtration studies showed that this ficolin-like protein also had a molecular weight of approx. 320 kDa under non-dissociating conditions. During the course of this study this ficolin-like protein was found to simply bind to CNBr-activated Sepharose which had been inactivated with Tris, and from which it could be eluted with GlcNAc. This ficolin-like protein was also shown to bind to GlcNAc, but not to mannose and maltose. The functional significance of the unusual binding property of this ficolin-like protein is not clear, but it has facilitated the development of a simple method for its purification. Keywords: Ficolin; P35; Fibrinogen-like; Collagen-like; GlcNAc
1. Introduction Ficolin is characterized by the presence of both collagen-like and fibrinogen-like sequences and is predicted to have either a ‘tulip-like’ or ‘cruciformAbbreviations: FBG, fibrinogen beta and gammarC-terminal; GlcNAc, N-acetyl-D-glucosamine; MBL, mannan binding lectin ) Corresponding author. Tel.: 65-7723679; Fax: 65-7721453.
like’ overall structure similar to those of complement protein C1q and the collectins ŽIchijo et al., 1993; Lu et al., 1993a; Holmskov et al., 1994.. Although porcine ficolin was initially isolated from the uterus as a membrane-associated, TGF-b 1-binding protein, human ficolin is synthesized mainly in peripheral blood by monocytes ŽLu et al., 1996a,b.. Using means of immunoblotting, a ficolin-related protein was also detected in human plasma as an E. coli-bi-
0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 0 2 9 - X
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Y. Le et al.r Journal of Immunological Methods 204 (1997) 43–49
nding protein which could be eluted from E. coli with sugars ŽLu et al., 1996b.. Recently, a novel protein, P35, purified from human serum was found to have a high degree of sequence identity to human ficolin. P35 was reported, like the collectins, to be a Ca2q-dependent lectin having a unique affinity for GlcNAc ŽMatsushita et al., 1996.. It was shown to be synthesized in the liver and to enhance phagocytosis of Salmonella typhimurium by polymorphonuclear leukocytes ŽMatsushita et al., 1996.. A single ficolin polypeptide consists of three regions, i.e. an N-terminal segment followed by a collagen-like sequence and then by a large C-terminal globular domain that is homologous to the Cterminal halves of fibrinogen b- and g-chains ŽIchijo et al., 1993; Lu et al., 1996a.. P35 is similar to human ficolin in domain organization and the two proteins also have 80% amino acid sequence identity ŽLu et al., 1996a; Matsushita et al., 1996.. The major structural difference between ficolin and P35 lies within the collagen-like regions where there is an apparent deletion of 12 residues or 4 Gly–Xaa–Yaa repeats in P35 ŽLu et al., 1996a; Matsushita et al., 1996.. The C-terminal globular domains in both ficolin and P35 contain putative Ca2q-binding siteŽs. and similar domains have been identified in a number of other proteins besides the b and g chains of fibrinogen, including the extracellular matrix proteins, tenascins ŽErickson, 1993.. This domain has been assigned the name of FBG ŽBork and Bairoch, 1995. which, in tenascin-C, has been shown to bind to heparin-Sepharose ŽFisher et al., 1995.. The presence of putative Ca2q-binding siteŽs. in the FBG domains of P35 is consistent with the fact that P35 has been reported to bind to GlcNAc in a Ca2q-dependent manner ŽMatsushita et al., 1996.. Independent studies have shown that proteins having identical N-terminal, or internal peptide, sequences to P35, i.e. hucolin and EBP-37, were also able to bind to cortisol and a-elastin ŽHarumiya et al., 1995; Edgar, 1996.. Therefore, ficolin and related proteins appear to bind to several apparently unrelated structures, i.e. TGF-b 1, cortisol, elastin, heparin and GlcNAc. Since human ficolin and P35 seem to belong to a closely related group of proteins, we tentatively assign the names ‘M-ficolin’ to the ficolin of monocyte origin and ‘L-ficolin’ to P35 to signify its synthesis in the liver.
In the present study, using human plasma as starting material we attempted to isolate the putative ficolin binding to E. coli by affinity chromatography on mannose-, maltose- and GlcNAc-Sepharose ŽLu et al., 1996b.. A protein present in the GlcNAc eluate from GlcNAc-Sepharose was found to have similar properties to L-ficolin. But this ficolin-like protein appeared to be distinct from L-ficolin in that its binding to GlcNAc was independent of Ca2q. A simple method has been developed for the purification of this ficolin-like protein based on its unusual affinity for CNBr-activated, but underivatized, Sepharose.
2. Materials and methods Q-Sepharose, collagenase Žtype VII., mouse antihuman IgA-, IgM-Sepharose, CNBr-activated agarose, divinyl sulfone and all sugars were obtained from Sigma Chemical Co. ŽSt. Louis, USA.. Sepharose 4B was purchased from Pharmacia Biotech ŽUppsala, Sweden. and derivatized with mannose, maltose or GlcNAc following the method of Fornstedt and Porath Ž1975.. Freshly frozen human plasma was obtained from the National University Hospital, Singapore. 2.1. Purification of ficolins from human plasma The methods for ficolin purification underwent modification after initial preparations were carried out using serum. Initially, serum was prepared from human plasma Ž1 l. and was first applied to an underivatized Sepharose 4B column Ž50 ml. for the purification of mannan binding lectin ŽTan et al., 1996.. The Sepharose adsorbed serum was then applied to a GlcNAc-Sepharose column Ž50 ml. and the column was, after washing with a TBS-TCa2q buffer Ž50 mM Tris, 150 mM NaCl, 0.05% Žwrv. NaN3 and 20 mM CaCl 2 , pH 7.8. Ž1 l., eluted first with TBS-TEDTA ŽTBS-TCa2q where CaCl 2 was replaced by 10 mM EDTA. and then with 100 mM GlcNAc in TBS-TEDTA. When a ficolin-like protein was detected in the GlcNAc eluate, but not in the EDTA eluate from the GlcNAc-Sepharose column Ždata not shown., it was decided that, in subsequent attempts to purify human ficolin, plasma rather than
Y. Le et al.r Journal of Immunological Methods 204 (1997) 43–49
serum would be used. The GlcNAc eluate containing the ficolin-like protein was passed through a Q-Sepharose column Ž1.5 = 10 cm. equilibrated with TBS-TEDTA to remove a major fraction of IgM and other proteins Ždata not shown.. The flow-through was diluted 3-fold with buffer D Ž50 mM Tris and 5 mM EDTA, pH 8.0. and applied to an FPLC mono-Q column. The column was eluted with a 50–450 mM NaCl gradient and the ficolin-like protein was eluted between 150–250 mM NaCl. The fractions containing the ficolin-like protein were pooled and applied to a CNBr-activated Sepharose column Ž5 ml. which had been blocked with Tris and the ficolin-like protein was eluted from this column pure with 100 mM GlcNAc in TBS-TEDTA. To determine the sugar specificity of the ficolin-like protein, human plasma Ž100 ml. was applied to a small CNBr-Sepharose column Ž5 ml.. The column was washed with TBSTEDTA and then sequentially eluted with 100 mM mannose, maltose and GlcNAc in TBS-TEDTA Ž10 = 1.0 mlreach elution.. The eluates were examined by SDS-PAGE. 2.2. SDS-PAGE and protein sequencing SDS-PAGE was carried out following the method of Laemmli Ž1970.. Protein samples were examined by SDS-PAGE either under reducing conditions Žincubation for 5 min at 958C in the presence on 50 mM dithiolthreitol. or under non-reducing conditions Žincubation of the samples in the presence of 100 mM iodoacetamide.. For protein sequencing, samples were separated by SDS-PAGE under reducing conditions and electroblotted onto Problott membranes, a polyvinylidene difluoride-type membrane with improved protein binding capacity ŽApplied Biosystems, Inc, Foster City, CA, USA.. Blotted proteins were visualized by Coomassie blue staining, excised and subjected to automated N-terminal sequencing on a 477 Ar120 A sequencer ŽApplied Biosystems.. 2.3. Collagenase digestion The purified ficolin-like protein Ž10 mg. was dialysed against 25 mM Tris ŽpH 7.4. containing 50 mM NaCl and 10 mM CaCl 2 . The dialysed samples were incubated for 24 h at 378C with collagenase Ž5 mg or
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10 units.. As controls, dialysed ficolin or collagenase alone were similarly incubated. The samples were then subjected to SDS-PAGE on a 5–15% Žwrv. gradient gel. 2.4. Gel-filtration The ficolin-like protein Žapprox. 100 mg in 0.2 ml. was subjected to HPLC gel-filtration on a Superose 6 column ŽHR 10r30. which had been equilibrated with TBS-TEDTA. The column was eluted at a flow rate of 0.3 mlrmin and the eluate was monitored at 280 nm ŽOD. for the elution profile. Eluted fractions were also examined by SDS-PAGE under reducing conditions. IgM, IgG and IgA were used as molecular weight standards.
3. Results 3.1. Purification of a ficolin-like protein In a previous study, a ficolin-related protein was immunochemically detected in serum as a putative lectin bound to carbohydrate structures on E. coli ŽLu et al., 1996b.. In the present study, attempts were made to isolate the protein from human serum by affinity chromatography on mannose-, maltoseand GlcNAc-Sepharose columns. A protein migrating as a band of approx. 40 kDa was detected in the GlcNAc eluates from GlcNAc-Sepharose columns. It was similar in size to the protein eluted from E. coli Ždata not shown. and had an N-terminal sequence identical to that of P35 or L-ficolin, over the first 14 residues ŽQAADTXPEVKMVGL; X denotes an undefined reside. ŽMatsushita et al., 1996.. A cortisolbinding protein identified in human serum has also been reported to have an identical N-terminal sequence to P35 or L-ficolin over the first 25 residues ŽEdgar, 1996.. Peptide sequences derived from an elastin-binding protein are also identical to L-ficolin ŽHarumiya et al., 1995.. Subsequent affinity isolation of this ficolin-like protein on GlcNAc-Sepharose showed that the protein examined in this study was bound to the column in a Ca2q-independent manner, since it was not eluted with EDTA, but was subsequently eluted with GlcNAc Ždata not shown.. This was in contrast to the reported Ca2q-dependent affin-
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Y. Le et al.r Journal of Immunological Methods 204 (1997) 43–49
Fig. 1. Elution of the ficolin-like protein from GlcNAc-Sepharose with GlcNAc. Human plasma Ž1 l. was applied to a GlcNAc-Sepharose Ž50 ml. column and bound proteins were eluted with 100 mM GlcNAc in TBS-TEDTA. Fractions Ž4 ml. were collected and examined by SDS-PAGE using a 10% Žwrv. gel under reducing conditions. The gel was stained with Coomassie blue. Lanes 1–11 denote continuous fractions of the GlcNAc elution.
ity of P35, or L-ficolin, for binding to GlcNAc-BSA ŽMatsushita et al., 1996.. Therefore, plasma rather than serum was used in the purification of the ficolin-like protein characterized in this study. The major contaminants in the GlcNAc eluate were immunoglobulins ŽFig. 1. as judged by immunoblotting with anti-human IgG, IgM and IgA antibodies Ždata not shown.. A major fraction of contaminating IgM was removed by passing the eluate, from the previous column, through a Q-Sepharose column equilibrated with TBS-TEDTA. The ficolin-like protein eluted in the flow-through from the Q-Sepharose was further purified on an FPLC mono-Q column from which it was eluted between 150–250 mM of the NaCl gradient. However, this preparation was still contaminated with IgM and IgA ŽFig. 2, lane 1.. To remove these immunoglobulins, the ficolin-like protein preparation eluted from mono-Q was applied to a mouse monoclonal anti-human IgA and IgM Žmouse IgG.-Sepharose column. Surprisingly, the ficolin-like protein as well as the contaminating IgA and IgM bound to the column although subsequent elution of the column with GlcNAc recovered only the ficolinlike protein in the eluate Ždata not shown.. The target structures bound by P35 or L-ficolin and various other proteins having the same N-terminal, or internal peptide, sequences are diverse since these targets include elastin-, cortisol- and IgG-Sepharose; the only common property of these affinity
resins being Sepharose. However, in one experiment, it was found that the ficolin-like protein did not bind to unactivated Sepharose Ždata not shown.. Therefore, a small column was prepared using CNBractivated agarose after inactivating the coupling sites with Tris. When the ficolin-like protein eluted from mono-Q ŽFig. 2, lane 1., which was still contaminated with immunoglobulins and other proteins, was applied to this CNBr-activated Sepharose column, only the ficolin-like protein bound to the column ŽFig. 2, lanes 1 and 2. and was subsequently recovered by elution with GlcNAc ŽFig. 2, lanes 3–11.. This unusual binding property of the ficolin-like protein means that it probably binds, non-specifically, to a wide range of columns prepared from CNBr-activated Sepharose and its binding properties revealed using immobilized ligands on CNBractivated Sepharose should be verified by other methods. The ficolin-like protein characterised in this study apparently bound to GlcNAc since, upon binding to GlcNAc- or CNBr-activated Sepharose, it could be eluted from the columns with GlcNAc in TBS-TEDTA. The affinity of porcine ficolin for TGF-b 1 has also been demonstrated in solution by cross-linking ŽIchijo et al., 1991.. Furthermore, hucolin is eluted from 11b-hydroxy-3-oxo-4-androstene-17b-carboxyaminoethylamine-1,4-butanediol
Fig. 2. Purification of the ficolin-like protein on CNBr-activated but underivatized Sepharose. CNBr-activated Sepharose was inactivated with 0.1 M Tris ŽpH 8.0. and fractions eluted from the mono-Q column containing the ficolin-like protein were pooled Žlane 1. and applied to the column Ž5 ml.. Lane 2 shows the flow-through. The column was, after washing with TBS-TEDTA, eluted with 100 mM GlcNAc. Fractions of the GlcNAc eluate Žlanes 3–11. were subjected to SDS-PAGE on a 10% Žwrv. gel under reducing conditions. The gel was stained with Coomassie blue.
Y. Le et al.r Journal of Immunological Methods 204 (1997) 43–49
Fig. 3. Disulphide-linked oligomerization of the ficolin-like protein. The purified protein was subjected to SDS-PAGE on a 5–15% Žwrv. gel under reducing Žlane 1. and non-reducing Žlane 2. conditions. The disulphide-linked oligomeric forms of purified human MBL ŽLu et al., 1990. were used as molecular weight standards Žlane 3.. The four bands in lane 3 are Žfrom the top. MBL hexamers Ž540 kDa., pentamers Ž450 kDa., tetramers Ž360 kDa. and trimers Ž270 kDa.. The MBL sample was a fraction eluted from a Sephacryl S-300 gel-filtration column ŽTan et al., 1996. and MBL trimer is under-represented in this fraction. The gel was stained with Coommassie blue.
diglycidylether-Sepharose with cortisol ŽEdgar, 1996. and EBP-37 binds to elastin immobilised on nitrocellulose ŽHarumiya et al., 1995.. While the explanation as to why the ficolin-like protein has an affinity for CNBr-activated Sepharose is not clear, it does seem clear that it is able to recognise structures on the CNBr-activated Sepharose which mimic the structure of GlcNAc. The unusual binding property of this ficolin-like protein has permitted the development of a simple method for its purification.
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Fig. 4. Molecular weight of the ficolin-like protein under non-dissociating conditions. The purified ficolin-like protein Žapprox. 100 mg in 0.2 ml. was applied to an FPLC Superose 6 column which had been equilibrated with TBS-TEDTA. The column was then eluted with the same buffer at a flow rate of 0.3 mlrmin and the eluate was monitored by absorbance at OD 280 . IgM, IgG and IgA were used as molecular weight standards. In the case of IgA, the elution position of dimers Žapprox. 350 kDa. is indicated.
Similar behaviour was also observed when porcine ficolin was subjected to SDS-PAGE ŽIchijo et al., 1991. and the 40 kDa polypeptide may represent a truncated form of the ficolin-like protein, similar to that reported for bovine conglutinin, due to partial degradation of the N-terminal region of the polypeptide ŽLu et al., 1993b.. The molecular weight of human ficolin was also examined under non-dissociating conditions by FPLC gel-filtration on a Superose 6 column. The ficolin-like protein was eluted immediately after dimeric IgA Žapprox. 350 kDa. as
3.2. Structural features of the ficolin-like protein The ficolin-like protein, resembled L-ficolin in that it migrated as a disulphide-linked oligomer of approx. 320 kDa on SDS-PAGE under non-reducing conditions ŽFig. 3, lane 2.. The molecular weight of the protein was judged by its migration between MBL trimers Žapprox. 270 kDa. and tetramers Žapprox. 360 kDa. ŽFig. 3, lane 3. ŽLu et al., 1990.. A low molecular weight species of ficolin was also observed under non-reducing conditions corresponding to a single polypeptide ŽFig. 3, lanes 1 and 2..
Fig. 5. Collagenase digestion of the ficolin-like protein. The purified ficolin-like protein Ž10 mg. was dialysed in a Ca2q-containing buffer Ž25 mM Tris, 50 mM NaCl and 10 mM CaCl 2 , pH 7.4. and then incubated at 378C for 24 h in the presence Žlanes 2. or absence Žlanes 3. of collagenase ŽSigma type VII; 5 mg or 10 units.. Collagenase alone Žlanes 1. was similarly incubated. The samples were subjected to SDS-PAGE on a 5–15% Žwrv. gradient gel under reducing conditions. The gel was silver–stained.
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a protein of approx. 320 kDa ŽFig. 4. showing that the ficolin-like protein underwent no further oligomerization beyond the 320 kDa disulphide-linked oligomer observed on SDS-PAGE ŽFig. 3, lane 2.. The molecular weight of the ficolin-like protein of 320 kDa indicates that it is likely to consist of fewer than 10 polypeptide chains and could contain 9 chains organized in three disulphide-linked triplehelices. The possible presence of a collagen-like sequence in the ficolin-like protein was confirmed by its sensitivity to collagenase. Digestion of ficolin with collagenase revealed a 32 kDa collagenase-resistent domain ŽFig. 5. which was considered to represent the C-terminal FBG domain since no other domain of similar size was expected when the collagen-like sequences in ficolin were degraded ŽLu et al., 1996a; Matsushita et al., 1996.. 3.3. Affinity of the ficolin-like protein for sugars To examine further the affinity of the ficolin-like protein for other sugars which were used in the elution of a ficolin-related protein from E. coli ŽLu et al., 1996b., human plasma Ž100 ml. was directly applied to a CNBr-activated Sepharose column.
Fig. 6. Sugar specificity of the ficolin-like protein. Human plasma Ž100 ml. was applied to a CNBr-activated Sepharose column Ž5 ml.. The CNBr-activated Sepharose was inactivated with 0.1 M Tris ŽpH 8.0. and equilibrated with TBS-TEDTA. Bound plasma proteins were eluted sequentially with 200 mM mannose, maltose and then GlcNAc in TBS-TEDTA and the eluates were subjected to SDS-PAGE on a 10% Žwrv. gel under reducing conditions. Lane 1, mannose eluate; lane 2, maltose eluate; lane 3, GlcNAc eluate. The gel was stained with Coomassie blue.
Bound proteins were eluted sequentially with mannose, maltose and GlcNAc. It was found that the 40 kDa ficolin-like protein was not present in the eluates obtained with mannose and maltose ŽFig. 6, lanes 1 and 2., but was subsequently eluted with GlcNAc ŽFig. 6, lane 3..
4. Discussion Porcine ficolin was isolated from uterus extract mainly by affinity chromatography on TGF-b 1-Sepharose ŽIchijo et al., 1991. and human L-ficolin, or P35, was affinity purified on a yeast mannan-Sepharose column and eluted from the column with GlcNAc ŽMatsushita et al., 1996.. We have previously showed that a human serum protein, immunologically related to M-ficolin, bound to E. coli Y1090 and was eluted from the bacteria with a mixture of three sugars, i.e. mannose, maltose and GlcNAc ŽLu et al., 1996b.. In the present study, we attempted to isolate M-ficolin and related proteins from human serum by affinity chromatography on mannose-, maltose- and GlcNAc-Sepharose columns. A ficolin-like protein has been characterized which appears to be identical to L-ficolin or P35 except that, unlike L-ficolin, it bound to GlcNAc in a Ca2q-independent manner. The relationship between this ficolin-like protein and L-ficolin is apparently close but cannot yet be clearly defined. Considering that the properties of the two proteins, i.e. N-terminal sequences, sizes of polypeptides, disulphide-linked oligomerization, presence of collagen-like sequences, and unique affinities for GlcNAc, are virtually identical except for Ca2q-dependence in GlcNAc binding, the two proteins may be identical, especially since L-ficolin was not found in the EDTA eluate from GlcNAcSepharose but the ficolin-like protein was subsequently eluted from the same column with GlcNAc Ždata not shown.. The affinity of the ficolin-like protein for CNBr-activated but underivatized Sepharose further implied similar binding properties to L-ficolin. L-ficolin was purified from human serum by affinity chromatography on mannan-Sepharose. Mannan is composed predominantly of high mannose structures but L-ficolin has been shown to bind to GlcNAc rather than mannose ŽNakajima and Bal-
Y. Le et al.r Journal of Immunological Methods 204 (1997) 43–49
lou, 1974; Matsushita et al., 1996.. This led to the suggestion that L-ficolin probably bound to GlcNAc-containing oligosaccharide contaminants on mannan-Sepharose, but it is also possible that Lficolin could simply have bound to the CNBractivated Sepharose resin to which mannan was attached. Whatever the explanation of the observed binding patterns, the affinity for CNBr-activated but underivatized Sepharose provided a simple method for the purification of the ficolin-like protein. The yield of the ficolin-like protein obtained based on this method has not been determined due to the lack of an assay for measuring its concentration in human plasma. However, a 50 ml GlcNAc-Sepharose column was shown to be sufficient to absorb most of the ficolinlike protein from 1 l human plasma because this protein was not detectable in such absorbed plasma when re-applied to a GlcNAc-Sepharose column Ždata not shown.. Approximately 300–400 m g of the ficolin-like protein can be purified by this method from 1 l plasma, as determined by a BCA Protein Assay ŽPierce, Rockford, USA.. Acknowledgements The authors wish to thank Dr. Maxey Chung for protein sequencing, Dahsila Dolmanan for technical assistance and Joseph Lau for photography. This work is supported by the National University of Singapore grant no. 940311. Yi Le is in receipt of a postgraduate scholarship of the National University of Singapore and J. Lu is concurrently in receipt of a Lee Kuan Yew Research Fellowship. References Bork, P., Bairoch, A., 1995. Extracellular protein modules. Trends. Biochem. Sci. 20, Appendix. Edgar, P.F., 1996. Hucolin, a new corticosteroid-binding protein from human plasma with structural similarities to ficolin, transforming growth factor-b 1-binding proteins. FEBS Lett. 375, 159. Erickson, H.P., 1993. Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions. Curr. Opin. Cell Biol. 5, 869. Fisher, D., Chiquet-Ehrismann, R., Bernasconi, C., Chiquet, M.,
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1995. A heparin binding region within the fibrinogen-like domain in chick tenascin-C. J. Biol. Chem. 270, 3378. Fornstedt, N., Porath, J., 1975. Characterization studies on a new lectin found in seeds of Vicia erÕilia. FEBS Lett. 57, 187. Harumiya, S., Omori, A., Sugiura, T., Fukumoto, Y., Tachkawa, H., Fujimoto, D., 1995. EBP-37, a new elastin-binding protein in human plasma: structural similarity to ficolins, transforming growth factor-b 1-binding proteins. J. Biochem. ŽTokyo. 117, 1029. Holmskov, U., Malhotra, R., Sim, R.B., Jensenius, J.-C., 1994. Collectins: collagen-like lectins of the innate immune system. Immunol. Today 15, 67. Ichijo, H., Ronnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., Miyazono, K., 1991. Purification of transforming growth factor-b 1 binding proteins from porcine uterus membranes. J. Biol. Chem. 33, 22459. Ichijo, H., Helman, U., Wernstedt, C., Gonez, L.J., ClaessonWelsh, I., Heldin, C.-H., Miyazono, K., 1993. Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen-like and collagen-like domains. J. Biol. Chem. 268, 14505. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature ŽLondon. 227, 680. Lu, J., Thiel, S., Wiedemann, H., Timpl, R., Reid, K.B.M., 1990. Binding of the pentamerrhexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2 C1s 2 complex, of the classical pathway of complement, without the involvement of C1q. J. Immunol. 144, 2287. Lu, J., Wiedemann, H., Timpl, R., Reid, K.B.M., 1993a. Structural similarity between C1q and the collectins as judged by electron microscopy. Behring. Inst. Mitt. 93, 6. Lu, J., Wiedemann, H., Holmskov, U., Thiel, S., Timpl, R., Reid, K.B.M., 1993b. Structural similarity between lung surfactant protein D and conglutinin, two distinct, C-type lectins containing collagen-like sequences. Eur. J. Biochem. 215, 793. Lu, J., Tay, P.N., Kon, O.L., Reid, K.B.M., 1996a. Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as the major site of synthesis and assignment of the gene to chromosome 9. Biochem. J. 313, 473. Lu, J., Le, Y., Kon, O.L., Chan, J., Lee, S.H., 1996b. Biosynthesis of human ficolin, an Escherichia coli-binding protein, by monocytes: comparison with the synthesis of two macrophage-specific proteins, C1q and the mannose receptor. Immunology 89, 289. Matsushita, M., Endo, Y., Taira, S., Sato, Y., Fujita, T., Ichikawa, N., Nakata, M., Mizuochi, T., 1996. A novel human lectin with collagen-like and fibrinogen-like domains which functions as an opsonin. J. Biol. Chem. 271, 2448. Nakajima, T., Ballou, C.E., 1974. Characterization of the carbohydrate fragments obtained from Saccharomyces cereÕisiae mannan by alkaline degradation. J. Biol. Chem. 249, 7679. Tan, S.M., Chung, M.C.M., Kon, O.L., Thiel, S., Lee, S.H., Lu, J., 1996. Improvements on the purification of human mannan binding lectin and demonstration of its Ca2q-independent association with a C1s-like protease. Biochem. J. 319, 329.
Purification and binding properties of a human ficolin-like protein Y. Le a , S.M. Tan a , S.H. Lee b, O.L. Kon a , J. Lu
a,)
a
b
Department of Biochemistry, Faculty of Medicine, National UniÕersity of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Department of Pathology, Faculty of Medicine, National UniÕersity of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 26 November 1996; revised 14 January 1997; accepted 10 February 1997
Abstract Ficolin was initially identified from porcine uterus as a TGF-b 1 binding protein and is considered to have an overall structure similar to that of the complement protein C1q and the collectins. Recent studies have shown that human ficolin is synthesized mainly by monocytes in peripheral blood and that it could potentially bind to sugar structures on microorganisms. The aim of the present investigations was to isolate ficolin from human plasma by affinity chromatography on immobilized sugars. A human serum protein was identified in the GlcNAc eluate from GlcNAc-Sepharose which migrated as a polypeptide of approx. 40 kDa on SDS-PAGE under reducing conditions and was, after further purification by FPLC on a mono-Q column, shown to have an identical N-terminal sequence, over the first 14 residues, to P35, a plasma protein having similar sequence and domain organisation to ficolin. This protein, named the ficolin-like protein, was shown to be sensitive to collagenase and similar to P35 in that it was also disulphide-linked into an oligomer of approx. 320 kDa. However, unlike P35, its binding to GlcNAc was independent of Ca2q. Gel-filtration studies showed that this ficolin-like protein also had a molecular weight of approx. 320 kDa under non-dissociating conditions. During the course of this study this ficolin-like protein was found to simply bind to CNBr-activated Sepharose which had been inactivated with Tris, and from which it could be eluted with GlcNAc. This ficolin-like protein was also shown to bind to GlcNAc, but not to mannose and maltose. The functional significance of the unusual binding property of this ficolin-like protein is not clear, but it has facilitated the development of a simple method for its purification. Keywords: Ficolin; P35; Fibrinogen-like; Collagen-like; GlcNAc
1. Introduction Ficolin is characterized by the presence of both collagen-like and fibrinogen-like sequences and is predicted to have either a ‘tulip-like’ or ‘cruciformAbbreviations: FBG, fibrinogen beta and gammarC-terminal; GlcNAc, N-acetyl-D-glucosamine; MBL, mannan binding lectin ) Corresponding author. Tel.: 65-7723679; Fax: 65-7721453.
like’ overall structure similar to those of complement protein C1q and the collectins ŽIchijo et al., 1993; Lu et al., 1993a; Holmskov et al., 1994.. Although porcine ficolin was initially isolated from the uterus as a membrane-associated, TGF-b 1-binding protein, human ficolin is synthesized mainly in peripheral blood by monocytes ŽLu et al., 1996a,b.. Using means of immunoblotting, a ficolin-related protein was also detected in human plasma as an E. coli-bi-
0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 0 2 9 - X
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nding protein which could be eluted from E. coli with sugars ŽLu et al., 1996b.. Recently, a novel protein, P35, purified from human serum was found to have a high degree of sequence identity to human ficolin. P35 was reported, like the collectins, to be a Ca2q-dependent lectin having a unique affinity for GlcNAc ŽMatsushita et al., 1996.. It was shown to be synthesized in the liver and to enhance phagocytosis of Salmonella typhimurium by polymorphonuclear leukocytes ŽMatsushita et al., 1996.. A single ficolin polypeptide consists of three regions, i.e. an N-terminal segment followed by a collagen-like sequence and then by a large C-terminal globular domain that is homologous to the Cterminal halves of fibrinogen b- and g-chains ŽIchijo et al., 1993; Lu et al., 1996a.. P35 is similar to human ficolin in domain organization and the two proteins also have 80% amino acid sequence identity ŽLu et al., 1996a; Matsushita et al., 1996.. The major structural difference between ficolin and P35 lies within the collagen-like regions where there is an apparent deletion of 12 residues or 4 Gly–Xaa–Yaa repeats in P35 ŽLu et al., 1996a; Matsushita et al., 1996.. The C-terminal globular domains in both ficolin and P35 contain putative Ca2q-binding siteŽs. and similar domains have been identified in a number of other proteins besides the b and g chains of fibrinogen, including the extracellular matrix proteins, tenascins ŽErickson, 1993.. This domain has been assigned the name of FBG ŽBork and Bairoch, 1995. which, in tenascin-C, has been shown to bind to heparin-Sepharose ŽFisher et al., 1995.. The presence of putative Ca2q-binding siteŽs. in the FBG domains of P35 is consistent with the fact that P35 has been reported to bind to GlcNAc in a Ca2q-dependent manner ŽMatsushita et al., 1996.. Independent studies have shown that proteins having identical N-terminal, or internal peptide, sequences to P35, i.e. hucolin and EBP-37, were also able to bind to cortisol and a-elastin ŽHarumiya et al., 1995; Edgar, 1996.. Therefore, ficolin and related proteins appear to bind to several apparently unrelated structures, i.e. TGF-b 1, cortisol, elastin, heparin and GlcNAc. Since human ficolin and P35 seem to belong to a closely related group of proteins, we tentatively assign the names ‘M-ficolin’ to the ficolin of monocyte origin and ‘L-ficolin’ to P35 to signify its synthesis in the liver.
In the present study, using human plasma as starting material we attempted to isolate the putative ficolin binding to E. coli by affinity chromatography on mannose-, maltose- and GlcNAc-Sepharose ŽLu et al., 1996b.. A protein present in the GlcNAc eluate from GlcNAc-Sepharose was found to have similar properties to L-ficolin. But this ficolin-like protein appeared to be distinct from L-ficolin in that its binding to GlcNAc was independent of Ca2q. A simple method has been developed for the purification of this ficolin-like protein based on its unusual affinity for CNBr-activated, but underivatized, Sepharose.
2. Materials and methods Q-Sepharose, collagenase Žtype VII., mouse antihuman IgA-, IgM-Sepharose, CNBr-activated agarose, divinyl sulfone and all sugars were obtained from Sigma Chemical Co. ŽSt. Louis, USA.. Sepharose 4B was purchased from Pharmacia Biotech ŽUppsala, Sweden. and derivatized with mannose, maltose or GlcNAc following the method of Fornstedt and Porath Ž1975.. Freshly frozen human plasma was obtained from the National University Hospital, Singapore. 2.1. Purification of ficolins from human plasma The methods for ficolin purification underwent modification after initial preparations were carried out using serum. Initially, serum was prepared from human plasma Ž1 l. and was first applied to an underivatized Sepharose 4B column Ž50 ml. for the purification of mannan binding lectin ŽTan et al., 1996.. The Sepharose adsorbed serum was then applied to a GlcNAc-Sepharose column Ž50 ml. and the column was, after washing with a TBS-TCa2q buffer Ž50 mM Tris, 150 mM NaCl, 0.05% Žwrv. NaN3 and 20 mM CaCl 2 , pH 7.8. Ž1 l., eluted first with TBS-TEDTA ŽTBS-TCa2q where CaCl 2 was replaced by 10 mM EDTA. and then with 100 mM GlcNAc in TBS-TEDTA. When a ficolin-like protein was detected in the GlcNAc eluate, but not in the EDTA eluate from the GlcNAc-Sepharose column Ždata not shown., it was decided that, in subsequent attempts to purify human ficolin, plasma rather than
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serum would be used. The GlcNAc eluate containing the ficolin-like protein was passed through a Q-Sepharose column Ž1.5 = 10 cm. equilibrated with TBS-TEDTA to remove a major fraction of IgM and other proteins Ždata not shown.. The flow-through was diluted 3-fold with buffer D Ž50 mM Tris and 5 mM EDTA, pH 8.0. and applied to an FPLC mono-Q column. The column was eluted with a 50–450 mM NaCl gradient and the ficolin-like protein was eluted between 150–250 mM NaCl. The fractions containing the ficolin-like protein were pooled and applied to a CNBr-activated Sepharose column Ž5 ml. which had been blocked with Tris and the ficolin-like protein was eluted from this column pure with 100 mM GlcNAc in TBS-TEDTA. To determine the sugar specificity of the ficolin-like protein, human plasma Ž100 ml. was applied to a small CNBr-Sepharose column Ž5 ml.. The column was washed with TBSTEDTA and then sequentially eluted with 100 mM mannose, maltose and GlcNAc in TBS-TEDTA Ž10 = 1.0 mlreach elution.. The eluates were examined by SDS-PAGE. 2.2. SDS-PAGE and protein sequencing SDS-PAGE was carried out following the method of Laemmli Ž1970.. Protein samples were examined by SDS-PAGE either under reducing conditions Žincubation for 5 min at 958C in the presence on 50 mM dithiolthreitol. or under non-reducing conditions Žincubation of the samples in the presence of 100 mM iodoacetamide.. For protein sequencing, samples were separated by SDS-PAGE under reducing conditions and electroblotted onto Problott membranes, a polyvinylidene difluoride-type membrane with improved protein binding capacity ŽApplied Biosystems, Inc, Foster City, CA, USA.. Blotted proteins were visualized by Coomassie blue staining, excised and subjected to automated N-terminal sequencing on a 477 Ar120 A sequencer ŽApplied Biosystems.. 2.3. Collagenase digestion The purified ficolin-like protein Ž10 mg. was dialysed against 25 mM Tris ŽpH 7.4. containing 50 mM NaCl and 10 mM CaCl 2 . The dialysed samples were incubated for 24 h at 378C with collagenase Ž5 mg or
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10 units.. As controls, dialysed ficolin or collagenase alone were similarly incubated. The samples were then subjected to SDS-PAGE on a 5–15% Žwrv. gradient gel. 2.4. Gel-filtration The ficolin-like protein Žapprox. 100 mg in 0.2 ml. was subjected to HPLC gel-filtration on a Superose 6 column ŽHR 10r30. which had been equilibrated with TBS-TEDTA. The column was eluted at a flow rate of 0.3 mlrmin and the eluate was monitored at 280 nm ŽOD. for the elution profile. Eluted fractions were also examined by SDS-PAGE under reducing conditions. IgM, IgG and IgA were used as molecular weight standards.
3. Results 3.1. Purification of a ficolin-like protein In a previous study, a ficolin-related protein was immunochemically detected in serum as a putative lectin bound to carbohydrate structures on E. coli ŽLu et al., 1996b.. In the present study, attempts were made to isolate the protein from human serum by affinity chromatography on mannose-, maltoseand GlcNAc-Sepharose columns. A protein migrating as a band of approx. 40 kDa was detected in the GlcNAc eluates from GlcNAc-Sepharose columns. It was similar in size to the protein eluted from E. coli Ždata not shown. and had an N-terminal sequence identical to that of P35 or L-ficolin, over the first 14 residues ŽQAADTXPEVKMVGL; X denotes an undefined reside. ŽMatsushita et al., 1996.. A cortisolbinding protein identified in human serum has also been reported to have an identical N-terminal sequence to P35 or L-ficolin over the first 25 residues ŽEdgar, 1996.. Peptide sequences derived from an elastin-binding protein are also identical to L-ficolin ŽHarumiya et al., 1995.. Subsequent affinity isolation of this ficolin-like protein on GlcNAc-Sepharose showed that the protein examined in this study was bound to the column in a Ca2q-independent manner, since it was not eluted with EDTA, but was subsequently eluted with GlcNAc Ždata not shown.. This was in contrast to the reported Ca2q-dependent affin-
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Fig. 1. Elution of the ficolin-like protein from GlcNAc-Sepharose with GlcNAc. Human plasma Ž1 l. was applied to a GlcNAc-Sepharose Ž50 ml. column and bound proteins were eluted with 100 mM GlcNAc in TBS-TEDTA. Fractions Ž4 ml. were collected and examined by SDS-PAGE using a 10% Žwrv. gel under reducing conditions. The gel was stained with Coomassie blue. Lanes 1–11 denote continuous fractions of the GlcNAc elution.
ity of P35, or L-ficolin, for binding to GlcNAc-BSA ŽMatsushita et al., 1996.. Therefore, plasma rather than serum was used in the purification of the ficolin-like protein characterized in this study. The major contaminants in the GlcNAc eluate were immunoglobulins ŽFig. 1. as judged by immunoblotting with anti-human IgG, IgM and IgA antibodies Ždata not shown.. A major fraction of contaminating IgM was removed by passing the eluate, from the previous column, through a Q-Sepharose column equilibrated with TBS-TEDTA. The ficolin-like protein eluted in the flow-through from the Q-Sepharose was further purified on an FPLC mono-Q column from which it was eluted between 150–250 mM of the NaCl gradient. However, this preparation was still contaminated with IgM and IgA ŽFig. 2, lane 1.. To remove these immunoglobulins, the ficolin-like protein preparation eluted from mono-Q was applied to a mouse monoclonal anti-human IgA and IgM Žmouse IgG.-Sepharose column. Surprisingly, the ficolin-like protein as well as the contaminating IgA and IgM bound to the column although subsequent elution of the column with GlcNAc recovered only the ficolinlike protein in the eluate Ždata not shown.. The target structures bound by P35 or L-ficolin and various other proteins having the same N-terminal, or internal peptide, sequences are diverse since these targets include elastin-, cortisol- and IgG-Sepharose; the only common property of these affinity
resins being Sepharose. However, in one experiment, it was found that the ficolin-like protein did not bind to unactivated Sepharose Ždata not shown.. Therefore, a small column was prepared using CNBractivated agarose after inactivating the coupling sites with Tris. When the ficolin-like protein eluted from mono-Q ŽFig. 2, lane 1., which was still contaminated with immunoglobulins and other proteins, was applied to this CNBr-activated Sepharose column, only the ficolin-like protein bound to the column ŽFig. 2, lanes 1 and 2. and was subsequently recovered by elution with GlcNAc ŽFig. 2, lanes 3–11.. This unusual binding property of the ficolin-like protein means that it probably binds, non-specifically, to a wide range of columns prepared from CNBr-activated Sepharose and its binding properties revealed using immobilized ligands on CNBractivated Sepharose should be verified by other methods. The ficolin-like protein characterised in this study apparently bound to GlcNAc since, upon binding to GlcNAc- or CNBr-activated Sepharose, it could be eluted from the columns with GlcNAc in TBS-TEDTA. The affinity of porcine ficolin for TGF-b 1 has also been demonstrated in solution by cross-linking ŽIchijo et al., 1991.. Furthermore, hucolin is eluted from 11b-hydroxy-3-oxo-4-androstene-17b-carboxyaminoethylamine-1,4-butanediol
Fig. 2. Purification of the ficolin-like protein on CNBr-activated but underivatized Sepharose. CNBr-activated Sepharose was inactivated with 0.1 M Tris ŽpH 8.0. and fractions eluted from the mono-Q column containing the ficolin-like protein were pooled Žlane 1. and applied to the column Ž5 ml.. Lane 2 shows the flow-through. The column was, after washing with TBS-TEDTA, eluted with 100 mM GlcNAc. Fractions of the GlcNAc eluate Žlanes 3–11. were subjected to SDS-PAGE on a 10% Žwrv. gel under reducing conditions. The gel was stained with Coomassie blue.
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Fig. 3. Disulphide-linked oligomerization of the ficolin-like protein. The purified protein was subjected to SDS-PAGE on a 5–15% Žwrv. gel under reducing Žlane 1. and non-reducing Žlane 2. conditions. The disulphide-linked oligomeric forms of purified human MBL ŽLu et al., 1990. were used as molecular weight standards Žlane 3.. The four bands in lane 3 are Žfrom the top. MBL hexamers Ž540 kDa., pentamers Ž450 kDa., tetramers Ž360 kDa. and trimers Ž270 kDa.. The MBL sample was a fraction eluted from a Sephacryl S-300 gel-filtration column ŽTan et al., 1996. and MBL trimer is under-represented in this fraction. The gel was stained with Coommassie blue.
diglycidylether-Sepharose with cortisol ŽEdgar, 1996. and EBP-37 binds to elastin immobilised on nitrocellulose ŽHarumiya et al., 1995.. While the explanation as to why the ficolin-like protein has an affinity for CNBr-activated Sepharose is not clear, it does seem clear that it is able to recognise structures on the CNBr-activated Sepharose which mimic the structure of GlcNAc. The unusual binding property of this ficolin-like protein has permitted the development of a simple method for its purification.
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Fig. 4. Molecular weight of the ficolin-like protein under non-dissociating conditions. The purified ficolin-like protein Žapprox. 100 mg in 0.2 ml. was applied to an FPLC Superose 6 column which had been equilibrated with TBS-TEDTA. The column was then eluted with the same buffer at a flow rate of 0.3 mlrmin and the eluate was monitored by absorbance at OD 280 . IgM, IgG and IgA were used as molecular weight standards. In the case of IgA, the elution position of dimers Žapprox. 350 kDa. is indicated.
Similar behaviour was also observed when porcine ficolin was subjected to SDS-PAGE ŽIchijo et al., 1991. and the 40 kDa polypeptide may represent a truncated form of the ficolin-like protein, similar to that reported for bovine conglutinin, due to partial degradation of the N-terminal region of the polypeptide ŽLu et al., 1993b.. The molecular weight of human ficolin was also examined under non-dissociating conditions by FPLC gel-filtration on a Superose 6 column. The ficolin-like protein was eluted immediately after dimeric IgA Žapprox. 350 kDa. as
3.2. Structural features of the ficolin-like protein The ficolin-like protein, resembled L-ficolin in that it migrated as a disulphide-linked oligomer of approx. 320 kDa on SDS-PAGE under non-reducing conditions ŽFig. 3, lane 2.. The molecular weight of the protein was judged by its migration between MBL trimers Žapprox. 270 kDa. and tetramers Žapprox. 360 kDa. ŽFig. 3, lane 3. ŽLu et al., 1990.. A low molecular weight species of ficolin was also observed under non-reducing conditions corresponding to a single polypeptide ŽFig. 3, lanes 1 and 2..
Fig. 5. Collagenase digestion of the ficolin-like protein. The purified ficolin-like protein Ž10 mg. was dialysed in a Ca2q-containing buffer Ž25 mM Tris, 50 mM NaCl and 10 mM CaCl 2 , pH 7.4. and then incubated at 378C for 24 h in the presence Žlanes 2. or absence Žlanes 3. of collagenase ŽSigma type VII; 5 mg or 10 units.. Collagenase alone Žlanes 1. was similarly incubated. The samples were subjected to SDS-PAGE on a 5–15% Žwrv. gradient gel under reducing conditions. The gel was silver–stained.
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a protein of approx. 320 kDa ŽFig. 4. showing that the ficolin-like protein underwent no further oligomerization beyond the 320 kDa disulphide-linked oligomer observed on SDS-PAGE ŽFig. 3, lane 2.. The molecular weight of the ficolin-like protein of 320 kDa indicates that it is likely to consist of fewer than 10 polypeptide chains and could contain 9 chains organized in three disulphide-linked triplehelices. The possible presence of a collagen-like sequence in the ficolin-like protein was confirmed by its sensitivity to collagenase. Digestion of ficolin with collagenase revealed a 32 kDa collagenase-resistent domain ŽFig. 5. which was considered to represent the C-terminal FBG domain since no other domain of similar size was expected when the collagen-like sequences in ficolin were degraded ŽLu et al., 1996a; Matsushita et al., 1996.. 3.3. Affinity of the ficolin-like protein for sugars To examine further the affinity of the ficolin-like protein for other sugars which were used in the elution of a ficolin-related protein from E. coli ŽLu et al., 1996b., human plasma Ž100 ml. was directly applied to a CNBr-activated Sepharose column.
Fig. 6. Sugar specificity of the ficolin-like protein. Human plasma Ž100 ml. was applied to a CNBr-activated Sepharose column Ž5 ml.. The CNBr-activated Sepharose was inactivated with 0.1 M Tris ŽpH 8.0. and equilibrated with TBS-TEDTA. Bound plasma proteins were eluted sequentially with 200 mM mannose, maltose and then GlcNAc in TBS-TEDTA and the eluates were subjected to SDS-PAGE on a 10% Žwrv. gel under reducing conditions. Lane 1, mannose eluate; lane 2, maltose eluate; lane 3, GlcNAc eluate. The gel was stained with Coomassie blue.
Bound proteins were eluted sequentially with mannose, maltose and GlcNAc. It was found that the 40 kDa ficolin-like protein was not present in the eluates obtained with mannose and maltose ŽFig. 6, lanes 1 and 2., but was subsequently eluted with GlcNAc ŽFig. 6, lane 3..
4. Discussion Porcine ficolin was isolated from uterus extract mainly by affinity chromatography on TGF-b 1-Sepharose ŽIchijo et al., 1991. and human L-ficolin, or P35, was affinity purified on a yeast mannan-Sepharose column and eluted from the column with GlcNAc ŽMatsushita et al., 1996.. We have previously showed that a human serum protein, immunologically related to M-ficolin, bound to E. coli Y1090 and was eluted from the bacteria with a mixture of three sugars, i.e. mannose, maltose and GlcNAc ŽLu et al., 1996b.. In the present study, we attempted to isolate M-ficolin and related proteins from human serum by affinity chromatography on mannose-, maltose- and GlcNAc-Sepharose columns. A ficolin-like protein has been characterized which appears to be identical to L-ficolin or P35 except that, unlike L-ficolin, it bound to GlcNAc in a Ca2q-independent manner. The relationship between this ficolin-like protein and L-ficolin is apparently close but cannot yet be clearly defined. Considering that the properties of the two proteins, i.e. N-terminal sequences, sizes of polypeptides, disulphide-linked oligomerization, presence of collagen-like sequences, and unique affinities for GlcNAc, are virtually identical except for Ca2q-dependence in GlcNAc binding, the two proteins may be identical, especially since L-ficolin was not found in the EDTA eluate from GlcNAcSepharose but the ficolin-like protein was subsequently eluted from the same column with GlcNAc Ždata not shown.. The affinity of the ficolin-like protein for CNBr-activated but underivatized Sepharose further implied similar binding properties to L-ficolin. L-ficolin was purified from human serum by affinity chromatography on mannan-Sepharose. Mannan is composed predominantly of high mannose structures but L-ficolin has been shown to bind to GlcNAc rather than mannose ŽNakajima and Bal-
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lou, 1974; Matsushita et al., 1996.. This led to the suggestion that L-ficolin probably bound to GlcNAc-containing oligosaccharide contaminants on mannan-Sepharose, but it is also possible that Lficolin could simply have bound to the CNBractivated Sepharose resin to which mannan was attached. Whatever the explanation of the observed binding patterns, the affinity for CNBr-activated but underivatized Sepharose provided a simple method for the purification of the ficolin-like protein. The yield of the ficolin-like protein obtained based on this method has not been determined due to the lack of an assay for measuring its concentration in human plasma. However, a 50 ml GlcNAc-Sepharose column was shown to be sufficient to absorb most of the ficolinlike protein from 1 l human plasma because this protein was not detectable in such absorbed plasma when re-applied to a GlcNAc-Sepharose column Ždata not shown.. Approximately 300–400 m g of the ficolin-like protein can be purified by this method from 1 l plasma, as determined by a BCA Protein Assay ŽPierce, Rockford, USA.. Acknowledgements The authors wish to thank Dr. Maxey Chung for protein sequencing, Dahsila Dolmanan for technical assistance and Joseph Lau for photography. This work is supported by the National University of Singapore grant no. 940311. Yi Le is in receipt of a postgraduate scholarship of the National University of Singapore and J. Lu is concurrently in receipt of a Lee Kuan Yew Research Fellowship. References Bork, P., Bairoch, A., 1995. Extracellular protein modules. Trends. Biochem. Sci. 20, Appendix. Edgar, P.F., 1996. Hucolin, a new corticosteroid-binding protein from human plasma with structural similarities to ficolin, transforming growth factor-b 1-binding proteins. FEBS Lett. 375, 159. Erickson, H.P., 1993. Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions. Curr. Opin. Cell Biol. 5, 869. Fisher, D., Chiquet-Ehrismann, R., Bernasconi, C., Chiquet, M.,
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1995. A heparin binding region within the fibrinogen-like domain in chick tenascin-C. J. Biol. Chem. 270, 3378. Fornstedt, N., Porath, J., 1975. Characterization studies on a new lectin found in seeds of Vicia erÕilia. FEBS Lett. 57, 187. Harumiya, S., Omori, A., Sugiura, T., Fukumoto, Y., Tachkawa, H., Fujimoto, D., 1995. EBP-37, a new elastin-binding protein in human plasma: structural similarity to ficolins, transforming growth factor-b 1-binding proteins. J. Biochem. ŽTokyo. 117, 1029. Holmskov, U., Malhotra, R., Sim, R.B., Jensenius, J.-C., 1994. Collectins: collagen-like lectins of the innate immune system. Immunol. Today 15, 67. Ichijo, H., Ronnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., Miyazono, K., 1991. Purification of transforming growth factor-b 1 binding proteins from porcine uterus membranes. J. Biol. Chem. 33, 22459. Ichijo, H., Helman, U., Wernstedt, C., Gonez, L.J., ClaessonWelsh, I., Heldin, C.-H., Miyazono, K., 1993. Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen-like and collagen-like domains. J. Biol. Chem. 268, 14505. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature ŽLondon. 227, 680. Lu, J., Thiel, S., Wiedemann, H., Timpl, R., Reid, K.B.M., 1990. Binding of the pentamerrhexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2 C1s 2 complex, of the classical pathway of complement, without the involvement of C1q. J. Immunol. 144, 2287. Lu, J., Wiedemann, H., Timpl, R., Reid, K.B.M., 1993a. Structural similarity between C1q and the collectins as judged by electron microscopy. Behring. Inst. Mitt. 93, 6. Lu, J., Wiedemann, H., Holmskov, U., Thiel, S., Timpl, R., Reid, K.B.M., 1993b. Structural similarity between lung surfactant protein D and conglutinin, two distinct, C-type lectins containing collagen-like sequences. Eur. J. Biochem. 215, 793. Lu, J., Tay, P.N., Kon, O.L., Reid, K.B.M., 1996a. Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as the major site of synthesis and assignment of the gene to chromosome 9. Biochem. J. 313, 473. Lu, J., Le, Y., Kon, O.L., Chan, J., Lee, S.H., 1996b. Biosynthesis of human ficolin, an Escherichia coli-binding protein, by monocytes: comparison with the synthesis of two macrophage-specific proteins, C1q and the mannose receptor. Immunology 89, 289. Matsushita, M., Endo, Y., Taira, S., Sato, Y., Fujita, T., Ichikawa, N., Nakata, M., Mizuochi, T., 1996. A novel human lectin with collagen-like and fibrinogen-like domains which functions as an opsonin. J. Biol. Chem. 271, 2448. Nakajima, T., Ballou, C.E., 1974. Characterization of the carbohydrate fragments obtained from Saccharomyces cereÕisiae mannan by alkaline degradation. J. Biol. Chem. 249, 7679. Tan, S.M., Chung, M.C.M., Kon, O.L., Thiel, S., Lee, S.H., Lu, J., 1996. Improvements on the purification of human mannan binding lectin and demonstration of its Ca2q-independent association with a C1s-like protease. Biochem. J. 319, 329.