ANALYSIS OF PROLINE RICH PROTEINS FROM HUMAN SALIVA

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。富含脯氨酸的蛋白质（PRP）是在人腮腺和下颂/舌下分泌物中大量存在的唾液蛋白质家族，并且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，它们被分为三组，酸性、碱性和糖基化的PRP。酸性PRP是一组8种结构上密切相关的分子，pI在4和5之间（Oppenheim等人，1971; Bennick和Connell，1971; Hay等人，1988年）。PIF、PRP1、PRP2含有150个残基，并且具有相同的氨基酸序列，除了在位置4和50处，其是天冬氨酸或天冬酰胺。PIFf、PRP3和PRP4由106个残基组成，分别与PIFs、PRP1和PRP2的N-末端106个残基相同。遗传学研究表明，PRP？爸？与PIF相同，除了在位置27的残基（其中亮氨酸取代异亮氨酸）和在位置103的残基（其中半胱氨酸取代精氨酸）（Azen等人，1987年）。另一个酸性PRP的特点是遗传分析？DB？含有PIF残基61 - 81的额外21个氨基酸重复的蛋白质，产生171个氨基酸残基的多肽链（Azen等，1987年）。酸性PRP表现出不寻常的生化特性。它们富含氨基酸甘氨酸、脯氨酸、谷氨酰胺和谷氨酸。氨基酸测序和化学分析表明，在PIF、PIFf、PRP 1、PRP 2、PRP 3和PRP 4中，残基8和22处的丝氨酸都被磷酸化。由于PRP的高度序列同源性和接近的pI值，这些蛋白质的分离需要具有极高分辨率的色谱法，并且通常涉及至少两个步骤，使用凝胶过滤和阴离子交换色谱技术的组合。这种纯化方案不仅麻烦，而且经常导致蛋白质污染和不希望的蛋白质损失。此外，这些程序使得在许多个体中定量不同的PRP是不切实际的。目前的调查已经导致了一个一步程序的发展，以分离的8个酸性PRP异构体和其特征在于的Pa和Db蛋白的一级结构和翻译后修饰。不久将提交报告这些结果的手稿。

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.