The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk - PubMed (original) (raw)

The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk

P Karhumaa et al. Proc Natl Acad Sci U S A. 2001.

Abstract

In addition to essential nutrients, human milk contains several classes of bioactive factors such as enzymes, hormones, and growth factors, many of which are implicated in infantile growth and development. Secretory carbonic anhydrase isoenzyme VI (CA VI) has been identified earlier as an essential component of mammalian saliva, and we demonstrate here by using biochemical and immunohistochemical techniques that it is also an elementary component of milk. The 42-kDa glycopolypeptide purified from human milk in CA inhibitor affinity chromatography shared 100% homology with salivary CA VI in the protein sequence analysis (40% coverage), and its digestion with PNGase F resulted in a polypeptide backbone similar in size to salivary CA VI. Quantification of CA VI in milk by using a time-resolved immunofluorometric assay revealed an approximately eight-times-higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum, in particular its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.

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Figures

Figure 1

(A) Western blot of the saliva of the human newborn infant (N saliva), human colostral milk (milk), human adult saliva (A saliva), and purified human salivary CA VI (CA VI) by using anti-human CA VI antibody (anti-CA VI) and normal rabbit serum (NRS). The anti-human CA VI antibody recognized the 42-kDa polypeptides of glycosylated CA VI in all of the samples. In addition, the 36-kDa polypeptide of the deglycosylated form of CA VI was visible in the purified human salivary CA VI sample. Control stainings using NRS were negative. (B) Western blot of rat milk (milk), rat mammary glands, and purified rat salivary CA VI (S CA VI) by using antibody raised against rat CA VI. All of the samples showed similar 42- and 36-kDa polypeptide bands. From three different mammary gland specimens, the positive signal was strongest in the lactating gland (lact.), moderate in the gland from pregnant animal (preg.), and faintest in the resting gland (rest.).

Figure 2

SDS/PAGE and colloidal Coomassie blue staining of total colostral milk (milk), CA purified from colostral milk (M CA VI; 0.2 μg, left lane; 0.6 μg, right lane), and CA purified from saliva (S CA VI). The ≈42-kDa polypeptides are seen in CAs purified from both milk and saliva. A polypeptide of similar size is also visible in the total milk sample.

Figure 3

PNGase F treatment of human salivary and milk CA VI followed by SDS/PAGE and Colloidal Coomassie blue staining. Without PNGase F treatment (−), the 42-kDa polypeptides for both salivary (S) and milk (M) CA VI are seen, corresponding to the glycosylated form of CA VI, but after digestion (+), the 36-kDa polypeptides for both samples are seen, indicating that the two glycopolypeptides have polypeptide cores of similar sizes.

Figure 4

Mean concentrations of CA VI in human colostrum and mature milk.

Figure 5

Immunohistochemical staining of rat mammary glands by using the anti-rat CA VI antibody (A, C, and_D_) and normal rabbit serum (NRS) (B). Milk inside the alveoli (arrows) stained strongly for CA VI in the lactating gland (A) and moderately in the gland of the pregnant animal (C). No staining was seen inside the alveoli of the resting gland (D). The cytoplasm of the alveolar epithelium showed a faint staining intensity (D, arrowheads) in all of the glands. Control staining of a lactating mammary gland with NRS was negative (B).

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The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk - PubMed (original) (raw)