Affinity and kinetic analysis of the bovine plasma C‐type lectin collectin‐43 (CL‐43) interacting with mannan

Holmskov, Uffe; Fischer, Per; Rothmann, Anette; Højrup, Peter

doi:10.1016/0014-5793(96)00915-5

Cited by 26 publications

(12 citation statements)

References 25 publications

(18 reference statements)

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“…This 66-kDa band was also detected by mass spectrometry (Holmskov et al, 1996) and could be a consequence of the purification process if the association of one chain is weaker than the others or if a small part of CL-43 exists as dimers. Reduced CL-43 is seen on SDSIPAGE as a main band at 43 kDa, together with a minor band approximately I-kDa smaller, corresponding to the truncated polypeptide lacking the first nine amino acid residues.…”

Section: Discussionmentioning

confidence: 90%

Structural Characterization of Bovine Collectin‐43

Rothmann¹,

Mortensen²,

Holmskov³

et al. 1997

European Journal of Biochemistry

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Bovine collectin-43 (CL-43), the most recently disclosed member of the collectin group, has been characterized structurally at the protein level by a combination of mass spectrometry and protein sequencing. The molecular mass of reduced CL-43 was determined by the use of mass spectrometry to be 33.6 2 0.1 kDa. Furthermore, the mass spectrum showed the presence of a truncated version of the polypeptide, which has also previously been shown by SDSPAGE and N-terminal sequencing. N-terminal Edman degradation of peptides from a tryptic digestion of native CL-43 verified the published sequence derived from cDNA studies and partial protein sequencing [Lim, B.-L., Willis, A. C., Reid, K. B. M., Lu, J., Lauersen, S. B., Jensenius, J. C. & Holmskov, U. (1994) J. Biol. Chem. 269, 11 820-11 8241 with two exceptions. Using mass spectrometry and N-terminal sequencing, a large number of post-translational modifications were found in the collagen-like region (repetitive Gly-Xaa-Yaa sequence). All proline residues located in the Yaa-position in the collagen-like region were found to be partially hydroxylated while all lysine residues in the Yaa position were fully hydroxylated and glycosylated. The glycosylation was determined as glycosyl-galactosyl 0-linked to a hydroxylated lysine residue. Mass spectrometric analysis of a peptic digest of the N-terminal tryptic peptide revealed that the three polypeptide chains were disulphide linked in a rather surprising pattern. The cysteine residues were inter-chain disulphide linked by Cysl5 in polypeptide chain 1 to Cysl5 in polypeptide chain 2, Cys20 in chain 2 to Cys20 in chain 3 and Cys20 in chain 1 to Cysl5 in chain 3.The four cysteine residues at the C-terminus were intra-chain disulphide linked, Cys204 to Cys299 and Cys277 to Cys291, as expected for a C-type lectin.Keywords: C-type lectin; mass spectrometry; post-translational modifications; disulfide bonds; collagenlike.Bovine CL-43 belongs to a group of proteins called collectins, containing a collagenous region linked to a C-terminal carbohydrate-recognition domain (CRD) common to the family of calcium-dependent carbohydrate-binding proteins known as C-type animal lectins (Drickamer, 1988). Other members of this family are bovine conglutinin and mannan-binding lectin (MBL), which are serum proteins like CL-43, and surfactant protein A and surfactant protein D (SP-A and SP-D), purified from lung pulmonary surfactant of various species. All collectins are involved in the innate immune defence. MBL, conglutinin, SP-D and SP-A has been shown to bind directly to polysaccharides on various pathogens, and it has been proposed that their collagenous regions are ligands for the collectin receptor on phagocytes, thus mediating phagocytosis of coated micro-organism (Malhotra et al., 1990). In addition, bovine conglutinin causes opsonization by binding to glycoproteins derived from complement C3, notably iC3b (Hirani et al., 1985;Lauersen et al., 1994;Friis-Christiansen et al., 1990). MBL also has the ability to activate the classical compl...

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Section: Discussionmentioning

confidence: 90%

Structural Characterization of Bovine Collectin‐43

Rothmann¹,

Mortensen²,

Holmskov³

et al. 1997

European Journal of Biochemistry

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“…It is more likely that a maximum of two Asn-846 oligomannose glycans are positioned within ϳ50 Å of each other and will bind two CRDs within one trimer. This would suggest that the K d of the fluid-phase interaction is Ͻ2-3 ϫ 10 Ϫ8 M (24). The lectin interaction between an MBL and fluid phase ␣2M is relatively weak and probably short-lived due to the low number of ligands (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…MBL binds to sugar residues via the CRDs (lectin) heads. (24). There are some human plasma proteins with which MBL does interact via oligomannose or GlcNAc terminating glycans such as has been described for specific glycoforms of IgG (IgG-G0) and IgM (25,26).…”

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confidence: 99%

Interaction of Mannan Binding Lectin with α2 Macroglobulin via Exposed Oligomannose Glycans

Arnold

Wallis

Willis

et al. 2006

Journal of Biological Chemistry

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The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor ␣ 2 macroglobulin (␣2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. ␣2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between ␣2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man 5-7 occupying Asn-846 on ␣2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active ␣2M (thiol ester intact) and proteasecleaved ␣2M (thiol ester cleaved). We demonstrate that MBL is able to interact with ␣2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to ␣2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral ␣2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.The protease inhibitor ␣ 2 macroglobulin (␣2M) 4 is a glycoprotein of 720 kDa, composed of four identical 180-kDa subunits, covalently linked in pairs by disulphide bridges. Each subunit has eight N-linked glycosylation sites located at Asn-32, Asn-47, Asn-224, Asn-373, Asn-387, Asn-846, Asn-968, and Asn-1401 (1) (see Fig. 1), a total of 32 N-linked glycosylation sites per ␣2M tetramer. ␣2M is an evolutionarily ancient protease inhibitor, which circulates in human plasma at 1-2 mg/ml and is found in a wide range of species from arthropods to mammals (2). There are two regions of major functional importance, a "bait region," which contains multiple cleavage sites for many different proteases (Fig. 1), and a second region, a reactive thiol ester (Fig. 1) also found in the complement components C3 and C4. ␣2M inhibits active proteases via a "trap mechanism" (3, 4), where cleavage of the "bait" region by the active protease triggers a conformational change in ␣2M (5) forming a cage-like structure around the protease. During the refolding, the protease or other "bystander" molecules (6) may become covalently bound via the thiol ester (7). The covalent binding, however, is not essential for protease inhibition (8). The entrapped protease is inhibited in its action on high molecular weight protein substrates, which can no longer access the cage. In contrast low molecular weight (sy...

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“…The modifications relative to 2 involved the removal of the platinum atom (9 and 11) and the removal of both the platinum atom and the pincer arms to yield an unsubstituted phenyl moiety (12). Free lactose and compounds 2, 9, 11, and 12 (1.25-40 mm) were allowed to flow across the RCA 120 surfaces to yield, after blank subtraction, the sensorgrams depicted in Figure 3 A-E.…”

Section: Unraveling the Characteristics Of The Organoplatinum(ii) Commentioning

confidence: 99%

“…Nevertheless, since the SPR response is proportional to the accumulation of mass on the sensor surface, a serious constraint imposed by this technique concerns the dimensions of the molecules to be employed as analytes. [7] In recent years, several groups have focused on SPR as an emerging technique to detect protein-carbohydrate interactions, [8][9][10][11][12][13][14][15][16][17][18][19][20] key steps in many biological events. [21,22] SPR studies of these biological events are hampered by the low availability of high-molecular-mass oligosaccharides and by the weakness of protein-carbohydrate interactions.…”

Section: Introductionmentioning

confidence: 99%

SPR Studies of Carbohydrate–Protein Interactions: Signal Enhancement of Low‐Molecular‐Mass Analytes by Organoplatinum(II)‐Labeling

et al. 2005

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The relatively insensitive surface plasmon resonance (SPR) signal detection of low‐molecular‐mass analytes that bind with weak affinity to a protein—for example, carbohydrate–lectin binding—is hampering the use of biosensors in interaction studies. In this investigation, low‐molecular‐mass carbohydrates have been labeled with an organoplatinum(II) complex of the type [PtCl(NCNR)]. The attachment of this complex increased the SPR response tremendously and allowed the detection of binding events between monosaccharides and lectins at very low analyte concentrations. The platinum atom inside the organoplatinum(II) complex was shown to be essential for the SPR‐signal enhancement. The organoplatinum(II) complex did not influence the specificity of the biological interaction, but both the signal enhancement and the different binding character of labeled compounds when compared with unlabeled ones makes the method unsuitable for the direct calculation of biologically relevant kinetic parameters. However, the labeling procedure is expected to be of high relevance for qualitative binding studies and relative affinity ranking of small molecules (not restricted only to carbohydrates) to receptors, a process of immense interest in pharmaceutical research.

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Affinity and kinetic analysis of the bovine plasma C‐type lectin collectin‐43 (CL‐43) interacting with mannan

Cited by 26 publications

References 25 publications

Structural Characterization of Bovine Collectin‐43

Structural Characterization of Bovine Collectin‐43

Interaction of Mannan Binding Lectin with α2 Macroglobulin via Exposed Oligomannose Glycans

SPR Studies of Carbohydrate–Protein Interactions: Signal Enhancement of Low‐Molecular‐Mass Analytes by Organoplatinum(II)‐Labeling

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