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The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.

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The complete amino acid sequence of a cellular retinol-binding protein (CRBP) has been determined for the first time. The primary structure of rat liver CRBP was elucidated by analyses of cyanogen bromide fragments and peptides obtained by tryptic and thermolytic digestions. The single polypeptide chain of rat CRBP consists of 134 amino acid residues. Under reducing conditions, CRBP exists as a monomer, but, in the absence of reducing agents, dimers and multimers of the protein emerge. This is explained by the observation that CRBP contains 3 cysteines, one of which seems to be highly reactive. Whether CRBP contains a disulfide bond is not yet established. The present data extend the previously described homology between CRBP and a family of low molecular weight proteins, all members of which may bind hydrophobic ligands. Since some of these proteins apparently display intracellular transport functions, a similar role for CRBP is envisaged.

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The primary structures of rabbit and rat prealbumin have been determined. The amino acid sequence of rabbit prealbumin was determined by analyses of peptides obtained by trypsin and Staphylococcus aureus protease digestions. The rat prealbumin sequence was deduced by analyses of tryptic peptides as well as by nucleotide sequencing of cDNA clones. Both amino acid sequences contain 127 amino acid residues, the same as human prealbumin. Pairwise comparisons show that the three sequences are more than 80% identical. All three prealbumins were found to display significant sequence homology with human thyroxine-binding globulin. A comparison of the primary structures of the prealbumins with the tertiary structure of human prealbumin shows that amino acid replacements are preferentially located at the surface of the molecule and in the loops connecting the beta-strands. The locations of the replacements are discussed as regards the different molecular interactions in which prealbumin is involved.

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Papain-solubilized human class II (HLA-DR) antigens have been purified from cadaveric spleens by ion-exchange chromatography, gel chromatography, and immunosorbent purification. The isolated papain-solubilized antigens comprised two subunits with apparent molecular weights of 23 000 and 30 000, respectively. The circular dichroism spectrum for the isolated class II antigens was similar to spectra recorded for HLA-A, -B, and -C antigens, immunoglobulins, and immunoglobulin fragments. Thus, class II antigens contain a considerable amount of beta structure. The small subunit (beta chain) exhibited extensive charge heterogeneity on two-dimensional isoelectric focusing polyacrylamide gel electrophoresis, whereas the large subunit (alpha chain) was more homogeneous. The structural heterogeneity of beta chains remained after neuraminidase treatment. The NH2-terminal amino acid sequence of the beta chains displayed multiple residues in several positions in accordance with the genetic polymorphism displayed by this chain. The alpha chain also displayed multiple residues in some positions, suggesting either that some of the genetic polymorphism of the class II antigens may be endowed in this chain or that multiple loci control the expression of several alpha chains. Papain-solubilized class II antigen subunits were homologous in their amino acid sequences with HLA-DR antigens of defined antigenic specificity as well as with murine I-E/C antigens.

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The stoichiometry of the interaction between prealbumin and retinol-binding protein was investigated. Gel chromatography analyses of prealbumin on columns equilibrated with retinol-binding protein (RBP)-containing buffers and fluorescence polarization analyses of RBP in the presence of various concentrations of prealbumin demonstrated that 3 molecules of RBP could simultaneously bind to prealbumin. Each RBP molecule seemed to interact with prealbumin with an apparent association constant of about 7.8 X 10(6) M-1. Fab fragments of anti-iudiotypic antibodies raised against anti-RBP antibodies reaced specifically with the RBP-binding sites on prealbumin. Two anti-idiotypic Fab fragments could simultaneously interact with prealbumin. These data strongly suggest that prealbumin exhibits at least two RBP-binding sites.

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Pooled, papain-solubilized HLA-A, -B, and -C antigens, derived from a large number of individuals and comprising several allelic forms, have been subjected to amino acid sequence determination. Despite the heterogeneity of the material, a main sequence representing all of the 273 amino acid residues could be established. The primary structure encompasses two immunoglobulin-like disulfide loops. The single carbohydrate moiety is attached to asparagine-86. Computer analyses demonstrated that the COOH-terminal one-third of the sequence, called H3, display statistically significant homology with members of the immunoglobulin family. The NH2-terminal two-thirds of the molecule, called H1 and H2, are not significantly homologous to any of the immunoglobulin sequences. However, H1 and H2 exhibit a distant relatedness to each other but no obvious similarity to the H3 region.

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The complete amino acid sequence of papain-solubilized HLA A, B and C antigens representing the cell surface-expressed portion of the transplantation antigens has been elucidated. The HLA antigen heavy chains seem to be composed of three domains, one of which binds beta 2-microglobulin. The HLA antigen heavy chain domain closest to the membrane displays striking amino acid homology with immunoglobulin light and heavy chains and with beta 2-microglobulin. The other two domains, which show no statistically significant homology with immunoglobulins, are related to each other. This suggest that the HLA antigen heavy chains have arisen by a series of gene duplication events.

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The classical human transplantation antigens, derived from the HLA-A, -B, and -C loci, are cell-surface-expressed glycoproteins. On the exterior of the cell the transplantation antigen heavy chain exposes two disulfide-containing domains and a glycosylated NH2-terminal extension. The disulfide-containing domain closest to the membrane has been isolated and its amino acid sequence has been determined. The HLA antigens used for the sequence analysis were derived from two and possibly three loci and comprised several allelic forms. The primary structure was remarkably invariant, and amino acid variations were observed only at three positions. Whether this suggests that the allelic variation of the HLA antigens is preferentially confined to other regions of the molecule or is a result of fortuitous selection of peptides remains to be established. The sequenced portion of the HLA antigen heavy chain is as homologous to beta 2-microglobulin and immunoglobulin light and heavy chains as are the latter to one another. This observation strengthens the notion that the transplantation antigens and the immunoglobulins are evolutionarily related.

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Papain-solubilized HLA-A, -B, and -C antigens have been isolated from cadaveric spleens. The isolated material was homogeneous and comprised subunits with the apparent molecular weights 33,000 and 12,000. Amino acid analyses of a mixture of HLA antigen heavy chains obtained from a great number of spleens with different HLA antigen phenotypes revealed a composition that is very similar to that of individual HLA-A and -B antigens. Likewise, the NH2-terminal 30 residues of the HLA-antigen heavy chain mixture were virtually identical with that recorded for individual specificities. The circular dichroism spectra for the isolated HLA antigens and for free beta2-microglobulin revealed similarities with spectra recorded for immunoglobulin chains and domains. The HLA-antigen heavy chain may contain an appreciable amount of beta structure. Antibodies raised against free beta2-microglobulin react better with beta2-microglobulin in free form than when bound to the HLA-A, -B, and -C antigen heavy chains. This is due to the fact that free beta2-microglobulin can bind a maximum of four Fab fragments simultaneously, whereas the HLA-antigen-associated beta2-microglobulin can bind only two Fab fragments without dissociating from the heavy HLA-antigen subunit.

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Highly purified, papain-solubilized HLA-A, -B, and -C antigens comprising a mixture of a great number of allelic forms from at least three loci have been fragmented by limited proteolysis, acid cleavage, and cyanogen bromide treatment. Limited proteolysis of 125I-labeled HLA-A, -B, and -C antigens with trypsin, chymotrypsin, thermolysin, and pepsin resulted in the production of two large fragments. One fragment was associated with beta 2-microglobulin and contained all of the carbohydrate. The other fragment, which had a molecular weight of about 13,000, is most probably derived from the COOH-terminal part of the heavy chain. Acid cleavage of the HLA antigen heavy chain gave rise to two main fragments with molecular weights of 22,000 and 11,000. Both fragments contained disulfide bonds. Two minor components, representing further cleavage products of the 22,000-dalton fragment, were also observed. Cleavage of the HLA antigen heavy chain at methionyl residues gave rise to one carbohydrate-containing, cysteine-free 14,000-dalton fragment and one 20,000-dalton fragment that contained all cysteines but no carbohydrate. NH2-terminal amino acid sequence analyses demonstrated that the 22,000-dalton acid cleavage fragment and the 14,000-dalton cyanogen bromide fragment were derived from the NH2-terminal part of the HLA antigen heavy chain.

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Milk fat globules (MFG), which are formed by exocytosis of lipid from epithelial cells of the mammary gland, are enveloped by plasma membrane from the epithelial cells. Highly purified, detergent-solubilized MFG membranes have been shown to contain molecules reactive with a rabbit antiserum against HLA-DR antigens. Indirect immunoprecipitation combined with polyacrylamide gel electrophoresis in sodium dodecyl sulfate demonstrated that the MFG membrane material reactive with the antiserum comprised molecules which under denaturing conditions displayed molecular weights of 28,000 and 35,000. The two types of polypeptide chains, which were both glycosylated, were held together by noncovalent forces under nondenaturing conditions. Various types of chemical and physicochemical analyses failed to reveal any significant differences between the HLA-DR-like antigens from MFG and from spleen cells. Since the HLA-DR-like antigens bound detergent in micellar form and were expressed on the outside of intact MFG, as revealed by indirect immunofluorescence, it is concluded that these antigens are embedded in the hydrocarbon matrix of the MFG and not merely passively adsorbed onto the MFG.

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Deoxycholate-solubilized HLA antigens have been isolated from platelets and comprised a mixture of 43,000- and 39,000-dalton polypeptide chains associated with beta2-microglobulin. Limited proteolysis experiments suggested that the 39,000-dalton chain is a fragment of the intact 43,000-dalton chain. Further proteolysis of the 39,000-dalton fragment yields a 33,000-dalton component. The 39,000-dalton molecule is more acidic than both the 43,000- and the 33,000-dalton chains. Differences in the amino acid compositions of the 43,000- and 39,000-dalton species demonstrate that the peptide(s) released on generation of the 39,000-dalton component are charged. The proteolytic split most probably occurs in the COOH-terminal end, which, owing to its content of charged amino acids, most probably is not integrated into the hydrocarbon matrix of the membrane. The 39,000- and 43,000-dalton components bind detergent in micellar form and can be incorporated into liposomes. The 33,000-dalton fragment has lost the ability to bind detergent micelles and is not incorporated into liposomes.

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The tentative amino acid sequence of pooled, papain-solubilized HLA antigen heavy chains has been determined. The amino acid sequence comprises 273 residues. As the structural analyses were performed on HLA antigen heavy chains comprising a mixture of several allelic forms derived from the A, B, and possibly C loci, multiple residues were encountered in several positions. However, a quantitatively dominating residue could always be easily identified. The present data suggest that the amino acid variability of the HLA-A, -B, and -C antigens is found in restricted regions of the molecule. The COOH-terminal third of the HLA antigen heavy chain appears to be less variable than other regions of the molecule. Previous work has shown that the HLA antigen heavy chain contains two immunoglobulin-like disulphide loops. The COOH-terminal third of the heavy chain was shown to be similar in primary structure to beta 2-microglobulin and the immunoglobulin G constant domains.

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A rabbit antiserum has been raised against highly purified, detergent-solubilized HLA-DR antigens. Externally or internally labelled surface glycoproteins from B-lymphocytes, epidermis, macrophages, and B-lymphoma cell lines reacted with the antiserum which precipitated polypeptide chains with the molecular weights 28,000 and 34,000. Such polypeptide chains were not observed when the antiserum was reacted with T-lymphocytes, T-cell lymphoma lines, kidney, brain, thymus and spermatozoa. Fab-fragments of the antiserum completely abolished the cytotoxic action of HLA-DR allantisera but had no effect on the cytotoxicity mediated by HLA-A,B and C loci antisera. Moreover, the rabbit antiserum reacted with the same molecules as the HLA-DR alloantisera. Fab-fragments of the rabbit antiserum completely abolished the MLR response and from the kinetics of the inhibition it is concluded that the Fab-fragments may interfere primarily with the initial recognition phase of the MLR.

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Papain-solubilized HLA-A,B,C antigen heavy chains have been cleaved by combined acid and CNBr treatment to yield three large fragments. A 14,000-dalton peptide comprises the NH2-terminal portion of the molecule, less a five-membered peptide. The 14,000-dalton fragment is followed in the linear sequence by a 9000-dalton peptide connected through an aspartyl-prolyl bond to the COOH-terminal 11,000-dalton fragment. The 9000- and 11,000-dalton fragments contain disulphide bridges that are immunoglobulin-like inasmuch as they encompass some fifty-five to sixty amino acid residues. The NH2-terminal portion of the HLA antigen heavy chain is devoid of cysteine. NH2-terminal amino acid sequence analyses do not reveal homologies between the 14,000- and 9000-dalton fragments, beta2-microglobulin, and the constant immunoglobulin domains. However, the NH2-terminal sequence of the 11,000-dalton fragment is as homologous to beta2-microglobulin and the constant immunoglobulin domains as they are to one another.

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