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First published online January 25, 2005
Journal of Experimental Biology 208, 487-496 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01398

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A distinct carbonic anhydrase in the mucus of the colon of humans and other mammals

Tanja Kleinke¹, Siegfried Wagner², Harald John³, David Hewett-Emmett⁴, Seppo Parkkila⁵, Wolf-Georg Forssmann³ and Gerolf Gros^*

¹ Zentrum Physiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
² Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
³ IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, D-30 625 Hannover, Germany
⁴ Human Genetics Center, University of Texas School of Public Health at Houston, Texas, USA
⁵ Institute of Medical Technology, University of Tampere and Tampere University Hospital, FIN-33014 Tampere, Finland

View larger version (22K): [in a new window]   Fig. 1. Distribution of CA activity after phase separation with Triton X-114. The values shown on the y axis are means ± S.D., from three experiments each for mucus of caecum and proximal colon from the guinea pig, two experiments for distal colon from the guinea pig, and four experiments for human red cell lysate and rabbit heart sarcolemmal vesicles. Values for heart sarcolemmal vesicles are from Bruns and Gros (1992).

View larger version (39K): [in a new window]   Fig. 2. Western blot analyses of human colonic mucus. For each analysis identical amounts of CA activity were applied for mucus samples and positive controls, respectively: 0.012 U ml-1 in the case of CAI, CAII and CAVI and 0.128 U ml-1 in the case of CAIV. Dilution of antibodies and detection were carried out as described in Materials and methods.

View larger version (56K): [in a new window]   Fig. 3. Western blot analysis of mucus CA from guinea pig. For each analysis identical amounts of CA activity applied with the samples were: guinea pig colonic mucus with 0.87 U ml-1 CA activity, isolated mucus CA with 5.3x10-3 U ml-1 CA activity. *Guinea pig CAI was applied at an amount of 0.87 U ml-1 CA activity as a positive control for guinea pig mucus. Applying CA I at 5.8x10-3 U ml-1 as a control for isolated a mucus CA did not result in a visible band (not shown).

View larger version (26K): [in a new window]   Fig. 4. Western blot analysis of mucus CA from guinea pig. Mucus from guinea pig caecum was applied at an activity amount of 0.87 U ml-1. Positive controls are (*) isolated guinea pig CA II at 5.1x10-3 U ml-1 CA activity and () isolated CA II at 0.79 U ml-1. Applying isolated mucus CA from the guinea pig at an activity amount of 5.3x10-3 U ml-1 did not result in a visible band.

View larger version (21K): [in a new window]   Fig. 5. Comparison of specific mucus CA activity in the caecum and colon of normal and germ-free rats. Means ± S.D. (N=5).

View larger version (31K): [in a new window]   Fig. 6. Comparison of partial sequences of isolated human mucus-CA with human CA I, CA II and relevant regions of human CA XIII and CA III. Partial sequences of human mucus-CA were obtained by Edman degradation of tryptic cleavage products separated by µHPLC. Alignment of the mucus-CA peptides was done in such a way as to maximize agreement with the CA I sequence. Numbering of the alignment is based on human CA I. Underlined sites show identity to the human mucus-CA peptide sequences. GenBank sequences used: human CA I (#P00915); human CA II (#P00918); human CA XIII (#NP_940986); human CA III (#P07451).

View larger version (21K): [in a new window]   Fig. 7. Evolutionary relationships of human mucus carbonic anydrase. Distances between protein sequences were not corrected for underestimated multiple mutations. Tree was built by the neighbor-joining method using a 68 amino acid alignment of the three human mucus CA peptides with selected CA isozyme sequences. The numbers on the branches represent the bootstrap replicates; only values above 75% are shown (for details see Hewett-Emmett and Tashian, 1996). GenBank protein sequence accessions: human CA I, II, III, XIII are given in the Fig. 6 legend; human CA IV (#P22748); CA VA (#NP_001730), CA VB (#NP_009151), CA VII (#P43166); Macaca nemestrina CA I (#P35217); mouse CA I (#NP_033929), CA II (#NP_033931), CA III (#NP_031632), CA IV (#P031633), CA VA (#NP_031634), CA VB (#NP_851832), CA VII (#NP_444300), CA XIII (#AAK16672); sheep CA I (#P48282); turtle CA I (cf. Table 1 in Hewett-Emmett and Tashian, 1996). *Bootstrap value <50%. 50%< bootstrap value <75%. Bold: bootstrap values >85% and support clustering of different CA genes.