lmmunocheraistry, 1975, Vol. 12, pp. 783-785. Pergamon Press, Printed in Great Britain
COMMUNICATION TO THE EDITORS HUMAN
SECRETORY COMPONENT--Ill
CARBOHYDRATES, AMINO ACIDS AND N-TERMINAL SEQUENCE K. SLETTEN*, T. B. C H R I S T E N S E N * and P. B R A N D T Z A E G t University of Oslo, Norway (First received 19 December 1974; in revised form 12 February 1975)
Abstract--Free secretory component purified from colostrum had a total carbohydrate content of 18.6 per cent by weight. Its amino acid composition showed lack of methionine, a relatively low content of the basic amino acids lysine and arginine, and a fairly high content of half cystine (3.3 residues per 100). Within the N-terminal amino acid sequence no heterogeneity was found, and there was no sequence homology with immunoglobulin light or heavy chains.
INTRODUCTION The secretory component (SC) is produced by serous epithelial cells of exocrine glands (Brandtzaeg, 1974a; Poger and Lamm, 1974). It occurs in the external secretions partly in a free form (Brandtzaeg, 1971b), partly associated with dimeric IgA (Tomasi et al., 1965) and pentameric IgM (Brandtzaeg, 1975), which both are selectively transmitted by the glandular cells (Brandtzaeg, 1971a). Since SC shows specific, non-covalent affinity for these two immunoglobufins in vitro (Brandtzaeg, 1974c), it has been suggested that the free component present in the membranes of secretory epithelial cells may act as a receptor protein responsible for the external immunoglobulin transport (Brandtzaeg, 1974a, b). It is therefore important to characterize this component to obtain information pertaining to its evolutionary origin and Ig-binding properties. Data in the literature show pronounced incongruity, probably because of the difficulties inherent in the purification of SC. This study is based on a preparation of free SC isolated by a new method (Brandtzaeg, 1974d) and subjected to extensive physicochemical characterization as reported elsewhere (Brandtzaeg, 1974e).
mg of lyophilized free SC was used for manual Edman degradation (Sletten et al., 1968). The phenylthiohydantoin amino acids were analyzed as described earlier (Sletten and Husby, 1974). A small amount of free SC was also used for the dansyl-Edman method (Gray, 1972). The dansyl amino acids were identified by thin-layer chromatography on polyamide sheets (Gray, 1972).
RESULTS AND DISCUSSION Carbohydrate analyses of SC showed a fairly high content of N-acetylglucosamine, but only trace amounts of N-acetylgalactosamine (Table I). The total carbohydrate content was found to be 18.6 per cent by weight. This is in the upper range of previously reported values, which are 9.5 per cent (Tomasi and Bienenstock, 1968), 15 per cent (Kobayashi, 1971), 11.6 per cent (van Munster et al., 1972), 11.5 per cent (Tomana et al., 1972), and 19"4 per cent (Lamm and Greenberg, 1972). The results obtained by the amino acid analyses disagree with some of the previous reports (Tomasi and Bienenstock, 1968; Tomana et al., 1972; Lamm and Greenberg, 1972; Mestecky et al., 1972), but are in strikingly good accordance with two recent studies based on extensively, MATERIALS AND METHODS although differently, purified preparations of free SC (Table Free SC was purified from colostrum by a combination 2). Lack of methionine seems to be an indicator of high of ammonium sulfate precipitation, gel filtration, anionic purity of SC. Its relatively high content of half cystine may exchange chromatography and immunosorbent (Brandt- explain the fact that after non-covalent interaction between zaeg, 1974d). Carbohydrate analyses were performed SC and dimeric IgA in vitro, a covalent stabilization of according to the method of Chambers and Clamp (1971). the complexes readily takes place, probably by disulfide The monosaccharides were released by methanolysis and, interchange (Brandtzaeg, 1974c). after re-N-acetylation, analyzed as the trimethyl silyl deriThe results from the N-terminal sequence analyses are vatives by gas chromatography. Amino acid analyses were shown in Table 3. In position 2, no PTH amino acid could carried out on a BioCal BC 200 Amino Acid Analyzer be detected in the ethylacetate fraction; neither tests perconnected to an Autolab Computing Integrator (System formed on the water-phase gave any clear answer. The AA) after acid hydrolysis of the samples in vacuum for yields of the PTH amino acids in positions 1, 3 and 4, 24 and 74 hr, as well as in 6N HC1 containing 0"05% thiog- expressed in nmol per 100 mg of free SC, were almost lycolic acid. Half cystine was analyzed as cysteic acid after identical in these steps (Table 3), and amounted to 15-207o performic acid oxidation of the sample (Hits, 1956). Sixteen of the starting material when based on a tool. wt of 83,000 (Brandtzaeg, 1974e). This yield is lower than that normally obtained with immunoglobulin light and heavy chains * Department of Biochemistry, Faculty of Mathematics (Hannestad and Sletten, 1971); but this could be due to and Natural Sciences. the high content of carbohydrate. A marker charge heterot Department of Microbiology, Dental Faculty and geneity has been observed by anionic exchange chromatImmunohistoehemical Laboratory, Institute of Pathology, ography and by isoelectric focusing of the free SC (BrandtRikshospitalet. zaeg, 1971b, 1974e). However, the N-terminal sequence 783
784
Communication to the Editors Table 1. Carbohydrate composition of free secretory component Weight percentage Fucose Mannose Galactose N-acetylglucosamine N-acetylgalactosamine N-acetylneuraminic acid
2'1 3.6 4.1 4"8 traces 4'0
Table 2. Amino acid composition of free secretory component (residues per 100) This report
Van Munster et al. (1972)
Kobayashi (1971)
10.7 5"6 8'0 11"0 5.3 9.8 6"1 3-3 8"7~ 0"0 3.9a 9.1 4"0 3"2 0-9 5"5 4.9 ND
10-6 5'9 8-1 10'3 4.8 9.4 6"1 3.5 8.1
COMMUNICATION TO THE EDITORS HUMAN
SECRETORY COMPONENT--Ill
CARBOHYDRATES, AMINO ACIDS AND N-TERMINAL SEQUENCE K. SLETTEN*, T. B. C H R I S T E N S E N * and P. B R A N D T Z A E G t University of Oslo, Norway (First received 19 December 1974; in revised form 12 February 1975)
Abstract--Free secretory component purified from colostrum had a total carbohydrate content of 18.6 per cent by weight. Its amino acid composition showed lack of methionine, a relatively low content of the basic amino acids lysine and arginine, and a fairly high content of half cystine (3.3 residues per 100). Within the N-terminal amino acid sequence no heterogeneity was found, and there was no sequence homology with immunoglobulin light or heavy chains.
INTRODUCTION The secretory component (SC) is produced by serous epithelial cells of exocrine glands (Brandtzaeg, 1974a; Poger and Lamm, 1974). It occurs in the external secretions partly in a free form (Brandtzaeg, 1971b), partly associated with dimeric IgA (Tomasi et al., 1965) and pentameric IgM (Brandtzaeg, 1975), which both are selectively transmitted by the glandular cells (Brandtzaeg, 1971a). Since SC shows specific, non-covalent affinity for these two immunoglobufins in vitro (Brandtzaeg, 1974c), it has been suggested that the free component present in the membranes of secretory epithelial cells may act as a receptor protein responsible for the external immunoglobulin transport (Brandtzaeg, 1974a, b). It is therefore important to characterize this component to obtain information pertaining to its evolutionary origin and Ig-binding properties. Data in the literature show pronounced incongruity, probably because of the difficulties inherent in the purification of SC. This study is based on a preparation of free SC isolated by a new method (Brandtzaeg, 1974d) and subjected to extensive physicochemical characterization as reported elsewhere (Brandtzaeg, 1974e).
mg of lyophilized free SC was used for manual Edman degradation (Sletten et al., 1968). The phenylthiohydantoin amino acids were analyzed as described earlier (Sletten and Husby, 1974). A small amount of free SC was also used for the dansyl-Edman method (Gray, 1972). The dansyl amino acids were identified by thin-layer chromatography on polyamide sheets (Gray, 1972).
RESULTS AND DISCUSSION Carbohydrate analyses of SC showed a fairly high content of N-acetylglucosamine, but only trace amounts of N-acetylgalactosamine (Table I). The total carbohydrate content was found to be 18.6 per cent by weight. This is in the upper range of previously reported values, which are 9.5 per cent (Tomasi and Bienenstock, 1968), 15 per cent (Kobayashi, 1971), 11.6 per cent (van Munster et al., 1972), 11.5 per cent (Tomana et al., 1972), and 19"4 per cent (Lamm and Greenberg, 1972). The results obtained by the amino acid analyses disagree with some of the previous reports (Tomasi and Bienenstock, 1968; Tomana et al., 1972; Lamm and Greenberg, 1972; Mestecky et al., 1972), but are in strikingly good accordance with two recent studies based on extensively, MATERIALS AND METHODS although differently, purified preparations of free SC (Table Free SC was purified from colostrum by a combination 2). Lack of methionine seems to be an indicator of high of ammonium sulfate precipitation, gel filtration, anionic purity of SC. Its relatively high content of half cystine may exchange chromatography and immunosorbent (Brandt- explain the fact that after non-covalent interaction between zaeg, 1974d). Carbohydrate analyses were performed SC and dimeric IgA in vitro, a covalent stabilization of according to the method of Chambers and Clamp (1971). the complexes readily takes place, probably by disulfide The monosaccharides were released by methanolysis and, interchange (Brandtzaeg, 1974c). after re-N-acetylation, analyzed as the trimethyl silyl deriThe results from the N-terminal sequence analyses are vatives by gas chromatography. Amino acid analyses were shown in Table 3. In position 2, no PTH amino acid could carried out on a BioCal BC 200 Amino Acid Analyzer be detected in the ethylacetate fraction; neither tests perconnected to an Autolab Computing Integrator (System formed on the water-phase gave any clear answer. The AA) after acid hydrolysis of the samples in vacuum for yields of the PTH amino acids in positions 1, 3 and 4, 24 and 74 hr, as well as in 6N HC1 containing 0"05% thiog- expressed in nmol per 100 mg of free SC, were almost lycolic acid. Half cystine was analyzed as cysteic acid after identical in these steps (Table 3), and amounted to 15-207o performic acid oxidation of the sample (Hits, 1956). Sixteen of the starting material when based on a tool. wt of 83,000 (Brandtzaeg, 1974e). This yield is lower than that normally obtained with immunoglobulin light and heavy chains * Department of Biochemistry, Faculty of Mathematics (Hannestad and Sletten, 1971); but this could be due to and Natural Sciences. the high content of carbohydrate. A marker charge heterot Department of Microbiology, Dental Faculty and geneity has been observed by anionic exchange chromatImmunohistoehemical Laboratory, Institute of Pathology, ography and by isoelectric focusing of the free SC (BrandtRikshospitalet. zaeg, 1971b, 1974e). However, the N-terminal sequence 783
784
Communication to the Editors Table 1. Carbohydrate composition of free secretory component Weight percentage Fucose Mannose Galactose N-acetylglucosamine N-acetylgalactosamine N-acetylneuraminic acid
2'1 3.6 4.1 4"8 traces 4'0
Table 2. Amino acid composition of free secretory component (residues per 100) This report
Van Munster et al. (1972)
Kobayashi (1971)
10.7 5"6 8'0 11"0 5.3 9.8 6"1 3-3 8"7~ 0"0 3.9a 9.1 4"0 3"2 0-9 5"5 4.9 ND
10-6 5'9 8-1 10'3 4.8 9.4 6"1 3.5 8.1
