Chen Y, Sharp A H, Hata K, Yunker A M R, Polo-Parada L, Landmesser L T, McEnery M W
Department of General Medical Sciences, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Neuroscience. 2007 Mar 30;145(3):981-96. doi: 10.1016/j.neuroscience.2006.12.060. Epub 2007 Feb 20.
Synthetic peptides of defined amino acid sequence are commonly used as unique antigens for production of antibodies to more complex target proteins. We previously showed that an affinity-purified, site-directed polyclonal antibody (CW90) raised against a peptide antigen (CNGRMPNIAKDVFTKM) anticipated to be specific to a T-type voltage-dependent Ca(2+) channel subunit identified recombinant rat alpha1I/Ca(V)3.3 and two endogenous mouse proteins distinct in their developmental expression and apparent molecular mass (neonatal form 260 kDa, mature form 190 kDa) [Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, McEnery MW (2003) Immunological characterization of T-type voltage-dependent calcium channel Ca(V)3.1 (alpha 1G) and Ca(V)3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117:321-335]. In the present study, we further characterize the biochemical properties of the CW90 antigens. We show for the first time that recombinant alpha1I/Ca(V)3.3 is modified by N-glycosylation. Using peptide:N-glycosidase F (PNGase F), an enzyme that removes polysaccharides attached at Asn residues, and endoneuraminidase-N (Endo-N), which specifically removes polysialic acid modifications, we reveal that differential glycosylation fully accounts for the large difference in apparent molecular mass between neonatal and adult CW90 antigens and that the neonatal form is polysialylated. As very few proteins are substrates for Endo-N, we carried out extensive analyses and herein present evidence that CW90 reacts with recombinant alpha1I/Ca(V)3.3 as well as endogenous neural cell adhesion molecule-180 (NCAM-180). We demonstrate the basis for CW90 cross-reactivity is a five amino acid epitope (AKDVF) present in both alpha1I/Ca(V)3.3 and NCAM-180. To extend these findings, we introduce a novel polyclonal anti-peptide antibody (CW678) that uniquely recognizes NCAM-180 and a new antibody (CW109) against alpha1I/Ca(V)3.3. Western blot analyses obtained with CW678, CW109 and CW90 on a variety of samples confirm that the endogenous CW90 signals are fully attributed to the two developmental forms of NCAM-180. Using CW678, we present novel data on differentiation-dependent NCAM-180 expression in human neuroblastoma IMR32 cells. These results strongly suggest the need for careful analyses to validate anti-peptide antibodies when targeting membrane proteins of low abundance.
具有特定氨基酸序列的合成肽通常用作独特抗原，以产生针对更复杂靶蛋白的抗体。我们之前表明，针对一种预计对T型电压依赖性Ca(2+)通道亚基具有特异性的肽抗原（CNGRMPNIAKDVFTKM）产生的亲和纯化的、位点定向的多克隆抗体（CW90）鉴定出重组大鼠α1I/Ca(V)3.3和两种内源性小鼠蛋白，它们在发育表达和表观分子量上有所不同（新生形式为260 kDa，成熟形式为190 kDa）[Yunker AM，Sharp AH，Sundarraj S，Ranganathan V，Copeland TD，McEnery MW（2003年）T型电压依赖性钙通道Ca(V)3.1（α1G）和Ca(V)3.3（α1I）亚型的免疫特征揭示了它们在定位、表达和神经发育方面的差异。神经科学117：321 - 335]。在本研究中，我们进一步表征了CW90抗原的生化特性。我们首次表明重组α1I/Ca(V)3.3被N - 糖基化修饰。使用肽：N - 糖基化酶F（PNGase F），一种去除连接在Asn残基上的多糖的酶，以及内切神经氨酸酶 - N（Endo - N），它特异性去除多唾液酸修饰，我们揭示差异糖基化完全解释了新生和成年CW90抗原之间表观分子量的巨大差异，并且新生形式是多唾液酸化的。由于很少有蛋白质是Endo - N的底物，我们进行了广泛分析，并在此提供证据表明CW90与重组α1I/Ca(V)3.3以及内源性神经细胞粘附分子 - 180（NCAM - 180）反应。我们证明CW90交叉反应性的基础是α1I/Ca(V)3.3和NCAM - 180中都存在的一个五氨基酸表位（AKDVF）。为了扩展这些发现，我们引入了一种独特识别NCAM - 180的新型多克隆抗肽抗体（CW678）和一种针对α1I/Ca(V)3.3的新抗体（CW109）。用CW678、CW109和CW90对各种样品进行的蛋白质印迹分析证实，内源性CW90信号完全归因于NCAM - 180的两种发育形式。使用CW678，我们提供了关于人神经母细胞瘤IMR32细胞中分化依赖性NCAM - 180表达的新数据。这些结果强烈表明，在针对低丰度膜蛋白时，需要仔细分析以验证抗肽抗体。
