ANALYSIS OF PROLINE RICH PROTEINS FROM HUMAN SALIVA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。富含脯氨酸蛋白(PRPS)是一类唾液蛋白家族，大量存在于人类腮腺和颌下/舌下分泌物中，占这些分泌物中蛋白质的10-40%(Kousvelari等人，1980；Baum等人，1982；Mandel&Bennick 1983)。根据它们的化学性质，它们被分为三类：酸性的、碱性的和糖基化的PRPS。酸性的PRPS是一组结构上密切相关的分子，其等电点在4到5之间(Oppenheim等人，1971；Bennick和Connell，1971；Hay等人，1988)。PIF、PRP1、PRP2含有150个残基，除第4位和第50位为天冬氨酸或天冬氨酸外，其余氨基酸序列相同。Piff、PRP3和PRP4分别由106个残基组成，分别与PIFs、PRP1和PRP2的106个残基相同。遗传学研究表明，PRP、PA、P3基因的序列。除27位的亮氨酸取代异亮氨酸和103位的半胱氨酸取代精氨酸外，其余均与PIFs相同(Azen等人，1987年)。另一种经遗传分析表征的酸性PRP是？含有额外21个氨基酸重复的PIF的第61-81个氨基酸残基的蛋白质，导致171个氨基酸残基的多肽链(Azen等人，1987年)。酸性的PRPS表现出不同寻常的生化特征。它们富含甘氨酸、脯氨酸、谷氨酰胺和谷氨酸。氨基酸序列和化学分析表明，在PIFs、Piff、PRP1、PRP2、PRP3和PRP4中，第8位和第22位的丝氨酸都被磷酸化。由于PRPS的高度序列同源性和接近的等电点，分离这些蛋白质需要极高分辨率的层析，通常至少需要两个步骤，使用凝胶过滤和阴离子交换层析技术相结合。这种提纯方案不仅繁琐，而且经常导致蛋白质污染和不必要的蛋白质损失。此外，这些程序使得量化一些个体的不同PRP是不切实际的。本研究建立了一种一步法分离8种酸性PRP亚型，并对PA和DB蛋白的一级结构和翻译后修饰进行了表征。报道这些结果的手稿将很快提交。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proline-rich proteins (PRPs) are a family of salivary proteins present in abundant amounts in human parotid and submandibular/sublingual secretions and account for 10-40% of the proteins in these secretions (Kousvelari et al., 1980; Baum et al., 1982; Mandel & Bennick 1983). On the basis of their chemical properties, they are divided into three groups, acidic, basic and glycosylated PRPs. The acidic PRPs are a group of eight structurally closely related molecules with pIs between 4 and 5 (Oppenheim et al., 1971; Bennick and Connell, 1971; Hay et al., 1988). PIFs, PRP1, PRP2 contain 150 residues and have the same amino acid sequence except at positions 4 and 50, which are either aspartic acid or asparagine. PIFf, PRP3 and PRP4 are composed of 106 residues and are identical with the N-terminal 106 residues of PIFs, PRP1 and PRP2, respectively. Genetic studies revealed that the sequence of PRP ?Pa? is identical to PIFs except for the residue at position 27 where leucine substitutes for isoleucine and the residue at position 103 where cysteine substitutes for arginine (Azen et al., 1987). Another acidic PRP characterized by genetic analysis was the ?Db? protein that would contain an extra 21 amino acid repeat of residues 61-81 of PIFs resulting in a 171-amino acid residue polypeptide chain (Azen et al., 1987). The acidic PRPs exhibit unusual biochemical characteristics. They are rich in the amino acids glycine, proline, glutamine and glutamic acid. Amino acid sequencing and chemical analysis has shown that both the serines at residues 8 and 22 are phosphorylated in PIFs, PIFf, PRP1, PRP2, PRP3 and PRP4. Due to the high degree of sequence homology and close pI values of PRPs, separation of these proteins requires chromatography with extremely high resolution and usually involves at least two steps, using a combination of gel filtration and anion-exchange chromatographic techniques. This purification scheme is not only cumbersome, but often results in protein contaminants and unwanted protein loss. Furthermore, these procedures make it impractical to quantitate different PRPs in a number of individuals. The present investigation has resulted in the development of a one step procedure to separate the 8 acidic PRP isoforms and characterized the primary structure and post-translational modifications of the Pa and Db proteins. A manuscript reporting these results will be submitted shortly.