Phosphoprotein amino acid sequence

Caseins were once defined as the phosphoproteins that precipitate from milk at pH 4.6 but are now defined by their amino acid sequences. In the elucidation of the primary structures of the caseins a French group has taken a prominent part (Ribadeau Dumas et al., 1975). The four polypeptide chains of bovine casein are the aSi-, as2-, P-, and K-caseins. Of these, the two as-caseins and K-casein are each phosphorylated to different extents and K-casein also exists in a number of glycosylated forms. 

Hemmings HC, Jr, Girault JA, Williams KR, LoPresti MB, Greengard P (1989) ARPP-21, a cyclic AMP-regulated phosphoprotein (Mr = 21,000) enriched in dopamine-innervated brain regions. Amino acid sequence of the site phosphorylated by cyclic AMP in intact cells and kinetic studies of its phosphorylation in vitro. J Biol Chem 264 7726-7733. 

A hydrophobic 10 kDa protein is also associated with PS II preparations [14], but its function is obscure. The protein is phosphorylated by a membrane-bound protein kinase [15] and the identity of this phosphoprotein has been the subject of much speculation [16]. It is clearly not the 9 kDa polypeptide of Cyt 6-559 or the 8 kDa proteolipid subunit of ATP synthase as shown by N-terminal amino acid sequence [14]. 

The first evidence of a cellular substrate of the insulin receptor kinase came from White et al. (1985b) who described a 185 kDa phosphoprotein in Fao hepatoma cells which was rapidly phosphorylated upon insulin stimulation. ppl85, recently renamed IRS-1, is a cytosolic protein with at least 30 potential serine/threonine- and 10 potential tyrosine-phosphorylation sites (Sun et al., 1991). Six of these tyrosine-phosphorylation sites lie in the amino acid sequence motif Tyr-Met-Xaa-Met which is recognized in its phosphorylated form by the SH2 (src homology 2) domain of the phosphatidylinositol 3-kinase (PI 3-kinase) (Sun et al., 1991). 

Phosphoproteins.— Details of the amino-acid sequences at the phosphorylation sites of two proteins involved in glycogen metabolism have been published, and they show unusual features. For example, there is an unusually high proportion of hydroxyl side-chains near the phosphoserine at one of the phosphorylation sites in glycogen synthetase, ... 

Although not reported here in detail, a striking feature emerges from the early work on phosphoproteins and peptones. Invariably, amino acid analjrses revealed that the portions of the peptide chains which contain the phosphorus—probably esterified to serine—always are rich in leucine, isoleucine, aspartic and glutamic acids (78, 79). As will be discussed later in this article, these observations and some recent results indicate that certain types of amino acid sequences recur in all phosphorus-containing proteins. 

One point of interest emerges from these experiments, namely, that ovalbumin is the second protein from which phosphoserine has been isolated. As in the case of the dipeptide, SerP.Glu, isolated from casein (38,77) ovalbumin also contains an amino acid sequence with phosphoserine adjacent to a dicarboxylic acid. Moreover, the close association of this amino acid with aspartic and glutamic acids, isoleucine and alanine in ovalbumin (22, 68) and casein (78-80) suggests the existence of a systematically recurring sequence in phosphoproteins. 

In an attempt to determine the points of attachment of the pepsin-phosphorus, Flavin succeeded in the isolation of three peptides Thr.SerP SerP.Glu and Thr.(Glu, SerP) (22). This evidence indicates that the single phosphate group of this protein is esterified in part to a serine residue. Moreover, the amino acid sequence Thr.SerP.Glu represents the third example in which the phosphoserine residue in a phosphoprotein is linked to a dicarboxylic acid. The second point of attachment of this phosphate diester, however, is still unknown. 

There is increasing evidence in the literature that amino acid sequences with phosphoserine adjacent to a dicarboxylic acid recur systematically in phosphoproteins. It, therefore, seems probable that certain principles must exist which determine the biosynthesis of such amino acid sequences and their subsequent incorporation into the protein molecule. 

Mature parasite-infected erythrocyte surface antigen (MESA) is a 250-300 kDa-fibrillar phosphoprotein encoded by a gene on chromosome 5 (Cooke et ah, 2001). It has a 19 amino acid sequence that binds to the membrane cytoskeletal protein 4.1 (Waller et ah, 2003). The precise function of MESA is not known but parasites that do not express MESA are viable and cytoadhere. 

Phosphatase treatment The phosphopeptides in the mixture can be identified by recording MS (usually MALDl-MS) spectra before and after the treatment with alkaline phosphatase or phosphoprotein phosphatase. A net mass differential of 80 Da is caused by the addition of a phosphate group to Ser, Thr or Tyr residue. A prior knowledge of the amino acid sequence of the peptide facilitates the identification of the phosphorylation site. 

The elemental composition of casein is not greatly different from that of wool (Table 10.13). The approximate amino acid compositions of each component phosphoprotein are listed in Table 10.14, their relative proportions in Table 10.12, and the casein amino acid sequences are indicated in Figures 10.22 through 10.25. These sequences are subject to minor variations particularly between animal species. Casein has numerous non-food applications (Chapter 12.17). 

Much has been written about the structure and properties of casein. Casein contains about 1% by weight of P and consists mainly of four phosphoproteins, which are designated as Oji, 0 2, P and k. Smaller quantities of other phosphoproteins (e.g. y casein -3%) are present, but some of these at least are breakdown products of the main varieties. Each of the four main varieties has a characteristic amino acid sequence (Chapter 10.2). All four varieties have genetic variants, however, and both the... 

Biosynthesis of the polypeptide chain is realised by a complicated process called translation. The basic polypeptide chain is subsequently chemically modified by the so-called posttranslational modifications. During this sequence of events the peptide chain can be cleaved by directed proteolysis, some of the amino acids can be covalently modified (hydroxylated, dehydrogenated, amidated, etc.) or different so-called prosthetic groups such as haem (haemoproteins), phosphate residues (phosphoproteins), metal ions (metal-loproteins) or (oligo)saccharide chains (glycoproteins) can be attached to the molecule by covalent bonds. Naturally, one protein molecule can be modified by more means. 

In the study of phosphoproteins, high-affinity antiphosphotyrosine (and other phosphorylated amino acid) antibodies can be used for phosphoprotein enrichment with peptide mass and sequence analyses are used to identify phosphorylation sites (40-43). 

Phosvitin is the principal phosphoprotein of egg yolk. Hen egg yolk phosvitin contains 10% phosphate by weight with 120 of the total 222 residues per mole (MW 35,500) phosphorylated. There are 119 residues of phosphoserine and one residue of phosphothreonine. About 5-7 of the total serine residues of phosvitin are not phosphorylated (97). A distinctive feature of the primary sequence of phosvitin is the repeating units of phosphoserine followed by a basic amino acid residue (72). This is illustrated by the following nine sequences of amino acids found in phosvitin ... 

Pspsin (EC 3.4.23.1) a protease in the stomach of all vertebrates with the exception of stomachless fish (e.g. carp). Purified P. shows maximal activity at pH 1-2, but in the stomach the optimal pH is 2-4. Above pH 6, P. is inactivated by denaturation. It preferentially catalyses hydrolysis of peptide bonds between two hydrophobic amino acids (Phe-Leu, Phe-Phe, Phe-TyrT With the exception of protamines, keratin, mucin, ovomucoid and other carbohydrate-rich proteins, most proteins are attacked by P. The products of P. action are peptone, i. e. mixtures of peptides in the M range 300-3,000. P. is a highly acidic (pi 1), single chain phosphoprotein (327 amino acid residues of known primary sequence, M, 34,500), which is released from its zymogen (pepsinogen, 42,500) by autocatalysis in the presence of hydrochloric acid. 

---